Methods for Purification of AAV Vectors by Anion Exchange Chromatography

ABSTRACT

The present disclosure provides methods for purifying a recombinant AAV (rAAV) vector from a solution by anion-exchange chromatography (AEX) to produce an eluate enriched for full capsids and depleted of empty capsids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 63/253,215, filed Oct. 7, 2021, U.S. Provisional Patent ApplicationNo. 63/217,194, filed Jun. 30, 2021, and U.S. Provisional PatentApplication No. 63/109,049, filed Nov. 3, 2020, the contents of each ofwhich are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created Oct. 15, 2021, isnamed PC072555A_Sequence_Listing_ST25.txt and is 1,048,576 bytes insize.

FIELD OF INVENTION

The present invention relates to the purification of AAV, and inparticular recombinant AAV (rAAV) vectors by anion exchangechromatography.

BACKGROUND

Gene therapy, using a recombinant AAV (rAAV) vector to deliver atherapeutic transgene, has the potential to treat a wide range ofserious diseases for which no cure, and in many cases, limited treatmentexists (Wang et al. (2019) Nature Reviews 18:358-378). Manufacturing ofgene therapy vectors is complex and requires specialized methods topurify the therapeutic rAAV vector from host cell impurities, and fromviral capsids that do not contain a complete vector genome encoding thetherapeutic transgene. In addition to development of a purificationmethod that produces a clinical grade rAAV vector composition of highpurity and with a good safety and efficacy profile, the purificationmethod must also be scalable to high volume rAAV production to meetpatient needs.

Ultracentrifugation using a cesium chloride gradient sedimentation is arobust method for removal of host cell protein and DNA, as well asseparation of viral capsids that are empty (i.e., that do not contain avector genome), partially packaged (also referred to as “intermediatecapsids” and which contain a partial vector genome and/ornon-transgene-related DNA) or fully packaged vectors (also referred toas “full capsids” and which contain a complete vector genome) (Burnhamet al. (2015) Hum. Gene Ther. Meth. 26:228-245). However, cesiumchloride gradient purification is laborious, time consuming and notamenable to large scale manufacturing.

Ultracentrifugation using an iodixanol gradient is less labor intensivebut generally results in vector yields of lower purity (Hermens et al.Hum. Gene Ther. (1999) Chromatographic methods including affinity and/orion exchange chromatography have proven useful for large-scaleproduction of clinical grade rAAV, including separation of empty viralcapsids from full rAAV vectors.

Empty capsids are produced by the host cells that produce and packagethe recombinant vector genome in the viral capsid. An excess of emptycapsids are produced relative to full vectors in most mammalianexpression systems, and various systems generate 1-30% full vectors(Penaud-Budloo et al. Molecular Therapy, Methods & Clinical Dev (2018)8:166-180). The production of empty capsids may be due to an imbalancein the ratio of plasmids encoding the transgene to that of the rep/capgenes. The presence of empty capsids in a drug product may cause anundesirable immune response and/or compete with the recombinant vectorsfor binding sites on target cells.

Certain anion-exchange chromatographic methods, employing acetatebuffers and resins such as POROS™ 50 HQ and Q-Sepharose XL, have beenused to separate empty capsids from rAAV2 vector pools by relying on theslightly less anionic character of the empty capsids as compared to fullvectors (U.S. Pat. No. 7,261,544; Qu et al. (2007) J. Virol. Meth.140(1):183-192). A similar approach used a combination of affinity andion exchange chromatography (IEX) and a 10 mM to 300 mM Tris acetategradient at pH 8 with POROS™ 50 HQ resin to enrich for full AAV vectorsof various serotypes (Nass et al. (2018) Molec. Thera. Meth. & Clin.Dev. 9:33-46). Other studies have identified buffers and conditionsuseful for chromatographic separation of empty capsids from full AAVvectors. For instance, Urabe determined that AAV1 material could bediluted with a Tris-HCl buffer comprising MgCl₂ and glycerol for load onan anion exchange chromatography (AEX) column and that solutionscomprising antichaotropic ions were effective elution buffers forseparation of the empty AAV1 capsids from full vectors (Urabe et al.(2006) Molec. Ther. 13(4):823-828). Others have described dilution(e.g., 50-fold) of affinity chromatography eluates and the use ofshallow gradient elution (e.g., 20 mM to 180 mM NaCl) from a monolithicsupport in AEX methods for the separation of empty capsids from full AAVvectors (US 2019-0002841; US 2019-0002842; US 2019-0002843; US2018-0002844). However, these methods also employ a high pH (9.8 to10.2) which can lead to deamidation and/or aggregation of rAAV vectorand may lead to a decrease in the therapeutic potency.

Processes using a combination of methods including, for example and inno particular order, tangential flow filtration (TFF) of a host cellsupernatant, precipitation of the capsid material (including rAAVvectors and empty capsids) using ammonium sulfate, AEX chromatographyand size-exclusion chromatography have been developed to separate therAAV from empty capsids (Tomono et al. (2018) Molec. Ther. Meth. Clin.Dev. 11:180-190).

There remains a need for methods for preparation of clinical grade rAAVvector (e.g., rAAV9) with optimal purity, potency and consistency. Thesemethods include the separation of rAAV comprising a vector genome withtherapeutic transgene from empty AAV capsid at a scale necessary to meetthe clinical need for treatment of disease (e.g., Duchenne MuscularDystrophy (DMD), Friedreich's Ataxia (FA)).

SUMMARY

The present disclosure provides an improved AEX method of purificationof rAAV vectors including, but not limited to the separation of fullrAAV vectors (e.g., rAAV9 vectors) from empty capsids. Such purifiedfull rAAV vectors are suitable for production of a drug product foradministration to a human subject, such as a subject with DMD. Thedisclosure also provides a novel method of preparation of achromatography eluate comprising rAAV vectors (e.g., from affinitychromatography) for further purification by AEX. The disclosure alsoprovides methods for regenerating an AEX stationary phase that allow thestationary phase to be used in multiple chromatography runs whilemaintaining the integrity of the process (e.g., successful purificationof rAAV vectors, the separation of full vectors from empty capsids)while reducing manufacturing costs.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following embodiments (E).

E1. A method of purifying an rAAV vector by AEX, the method comprising astep of:

-   -   i) loading a solution comprising the rAAV vector to be purified        onto a stationary phase in a column;    -   ii) performing gradient elution of material from the stationary        phase in the column wherein a percentage of a first gradient        elution buffer is varied in a manner inversely proportional to        variation in a percentage of a second gradient elution buffer;    -   iii) collecting at least one fraction of eluate from the column        during the gradient elution beginning when the absorbance of a        column flow-through reaches an absorbance threshold.        E2. The method of E1, wherein loading the solution comprising        the rAAV vector onto the column comprises application of        2.5×10¹⁵ to 3.0×10¹⁷ vector genome (VG)/L of column volume onto        the column.        E3. The method of E1 or E2, wherein loading comprises        application of 8.0×10¹² to 2.0×10¹⁸ total VG to the column.        E4. The method of any one of E1-E3, wherein loading comprises        application of a diluted, and optionally filtered solution        comprising 2.6×10¹² to 6.8×10¹³ VG/mL of column volume onto a        column (e.g., a 6.4 L column) as measured by qPCR analysis of a        transgene sequence within the vector genome.        E5. The method of any one of E1-E4, wherein loading comprises        application of a diluted, and optionally filtered solution        comprising 5×10¹³ to 1.3×10¹⁴ VG/mL of column volume onto a        column (e.g., a 1.3 L column) as measured by qPCR analysis of        the ITR sequences within the vector genome.        E6. The method of any one of E1-E5, wherein the rAAV vector is        produced in a vessel, and wherein the volume of the vessel is        about 1 L, about 50 L, about 100 L, about 250 L, about 500 L,        about 1000 L, about 2000 L or greater.        E7. The method of any one of E1-E6, wherein the vessel is a        single use bioreactor (SUB).        E8. The method of any one of E1-E7, wherein the solution        comprising the rAAV vector is selected from the group consisting        of an affinity eluate, a supernatant from a cell lysate and a        post-harvest solution that has been diluted and optionally        filtered prior to loading.        E9. The method of any one of E1-E8, wherein the solution        comprising the rAAV vector is an affinity eluate that has been        diluted and optionally filtered prior to loading.        E10. The method of E1-E9, wherein the solution has undergone at        least one other purification or processing step.        E11. The method of E10, wherein the at least one other        purification or processing step is selected from the group        consisting of cell lysis, flocculation, filtration,        chromatography (e.g., affinity chromatography), dilution, pH        adjustment, conductivity adjustment and a combination thereof.        E12. The method of any one of E1-E12, wherein the solution        comprising the rAAV vector is an affinity eluate resulting from        affinity chromatography purification of a rAAV vector produced        in a single use bioreaction (SUB) with a volume.        E13. The method of E12, wherein the SUB has a volume of about 1L        to about 2000L.        E14. The method of any one of E1-13, wherein the stationary        phase is an AEX stationary phase.        E15. The method of any one of E1-E14, wherein the stationary        phase is positively charged.        E16. The method of any one of E1-E15, wherein the stationary        phase is a polystyrenedivinylbenzene particle with covalently        bound quaternized polyethyleneimine, and optionally wherein the        stationary phase is POROS™ 50 HQ.        E17. The method of any one of E1-E16, further comprising        application of a load chase solution to the AEX stationary phase        in the column, optionally after loading the solution comprising        the rAAV vector onto the stationary phase.        E18. The method of E17, wherein 1 to 15 column volumes (CV) of        the load chase solution comprising a buffering agent (e.g.,        Tris, BIS-Tris propane, diethanolamine, diethylamine, tricine,        triethanolamine and/or bicine) are applied to the stationary        phase in the column.        E19. The method of E17 or E18, wherein the load chase solution        comprises about 10 mM to 30 mM (e.g., about 20 mM) Tris, pH        8-10.        E20. The method of any one of E1-E19, further comprising pre-use        flushing of a stationary phase in a column.        E21. The method of E20, wherein the pre-use flushing of the        stationary phase in the column precedes loading the solution        comprising the rAAV vector onto the column.        E22. The method of E20 or E21, wherein the pre-use flushing of        the stationary phase in the column comprises application of        water for injection to the stationary phase.        E23. The method of any one of E1-E22, further comprising        sanitizing the stationary phase in the column.        E24. The method of E23, wherein sanitizing the stationary phase        in the column precedes loading the solution comprising the rAAV        vector onto the column.        E25. The method of E23 or E24, wherein sanitizing the stationary        phase in the column comprises application of a solution        comprising NaOH to the stationary phase.        E26. The method of any one of E23-E25, wherein sanitizing the        stationary phase in the column comprises application of a        solution comprising about 0.1 M to about 1.0 M (e.g., about        0.5 M) NaOH to the stationary phase.        E27. The method of any one of E23-E26, wherein sanitizing the        stationary phase in the column comprises application of about 5        CV to about 10 CV, or about 14.4 CV to about 17.6 CV of the        solution comprising about 0.1 M to about 1.0 M NaOH to the        stationary phase.        E28. The method of any one of E23-E27, wherein sanitizing the        stationary phase in the column is by upward flow.        E29. The method of any one of E1-E28, further comprising        regenerating the stationary phase in the column.        E30. The method of E29, wherein regenerating the stationary        phase in the column precedes loading the solution comprising the        rAAV vector onto the column.        E31. The method of E29 or E30, wherein regenerating the        stationary phase in the column comprises application of a        solution comprising a component selected from the group        consisting of a salt, a buffering agent and a combination        thereof to the stationary phase.        E32. The method of any one of E29-E31, wherein regenerating the        stationary phase in the column comprises application of a        solution comprising about 1 M to about 3 M (e.g., about 2 M)        NaCl, about 50 mM to about 150 mM (e.g., about 100 mM) Tris, pH        8 to 10 (e.g., about 9) to the stationary phase.        E33. The method of any one of E29-E32, wherein regenerating the        stationary phase in the column comprises application of a        solution comprising about 50 mM to about 150 mM (e.g., about 100        mM) Tris, pH 9 to the stationary phase.        E34. The method of any one of E29-E33, wherein regenerating the        stationary phase in the column comprises application of 4.5 to        5.5 CV of the solution comprising about 100 mM Tris, pH 9 to the        stationary phase.        E35. The method of any one of E29-E34, wherein regenerating the        stationary phase in the column is performed more than once.        E36. The method of any one of E1-E35, further comprising        equilibration of the stationary phase in the column.        E37. The method of E36, wherein equilibration of the stationary        phase in the column precedes or follows loading the solution        comprising the rAAV vector onto the column.        E38. The method of E36 or E37, wherein equilibration of the        stationary phase in the column comprises application of an        equilibration buffer comprising at least one component selected        from the group consisting of a buffering agent, a salt, an amino        acid, a detergent and a combination thereof, to the stationary        phase.        E39. The method of E38, wherein the buffering agent is selected        from the group consisting of Tris, BIS-Tris propane,        diethanolamine, diethylamine, tricine, triethanolamine and/or        bicine and a combination thereof.        E40. The method of E36 or E39, wherein the salt is selected from        the group consisting of sodium chloride, sodium acetate,        ammonium acetate, magnesium chloride, sodium sulfate and a        combination thereof.        E41. The method of any one of E38-E40, wherein the salt is        sodium acetate.        E42. The method of any one of E38-E41, wherein the amino acid is        selected from the group consisting of histidine, arginine,        glycine, citrulline and a combination thereof.        E43. The method of any one of E38-E42, wherein the amino acid is        histidine.        E44. The method of E38-E43, wherein the detergent is selected        from the group consisting of poloxamer 188 (P188), Triton X-100,        polysorbate 80 (PS80), Brij-35, nonyl phenoxypolyethoxylethanol        (NP-40) and a combination thereof.        E45. The method of any one of E38-E44, wherein the detergent is        P188.        E46. The method of any one of E36-E45, wherein equilibration of        the stationary phase in the column comprises application of an        equilibration buffer comprising about 50 mM to about 150 mM        (e.g., about 100 mM Tris), pH about 8 to 10 (e.g., about 9) to        the stationary phase.        E47. The method of any one of E36-E46, wherein equilibration of        the stationary phase in the column comprises application of an        equilibration buffer comprising about 50 mM to about 150 mM        (e.g., about 100 mM Tris), about 250 mM to about 750 mM (e.g.,        about 500 mM) sodium acetate, about 0.005 to about 0.015% (e.g.,        about 0.01%) P188, pH about 8 to about 10 (e.g., about 8.9) to        the stationary phase.        E48. The method of any one of E36-E47, wherein equilibration of        the stationary phase in the column comprises application of an        equilibration buffer comprising about 100 mM to about 300 mM        (e.g., about 200 mM) histidine, about 100 mM to about 300 mM        (e.g., about 200 mM Tris), about 0.1% to about 1.0% (e.g., about        P188, pH about 8 to about 10 (e.g., about 8.8) to the stationary        phase.        E49. The method of any one of E36-E48, wherein equilibration of        the stationary phase in the column comprises application of an        equilibration buffer comprising about 50 mM to about 150 mM        (e.g., about 100 mM) Tris, 0.005% to about 0.015% (e.g., about        0.01%) P188, pH about 8 to about 10 (e.g., about 8.9) to the        stationary phase.        E50. The method of any one of E36-E49, wherein equilibration of        the stationary phase in the column comprises application of        greater than about 4.5 CV of an equilibration buffer to the        stationary phase.        E51. The method of any one of E36-E50, wherein equilibration of        the stationary phase in the column comprises application of 4.5        to 5.5 CV of an equilibration buffer to the stationary phase.        E52. The method of any one of E36-E51, wherein equilibration of        the stationary phase in the column is performed more than once.        E53. The method of any one of E36-E52, wherein at least one        equilibration buffer is applied to the stationary phase prior to        loading the solution comprising the rAAV vector onto the column;        and wherein at least one equilibration buffer is applied to the        stationary phase after loading the solution comprising the rAAV        vector onto the column.        E54. The method of any one of E1-E53, comprising performing        gradient elution of material from the stationary phase in the        column.        E55. The method of E54, wherein the gradient elution comprises        application of 10 to 60 CV of at least two different solutions        (e.g., gradient elution buffers), or a mixture of the two, to        the stationary phase, and wherein over the course of the        gradient elution, a percentage of a first solution is varied in        a manner inversely proportional to a percentage of a second        solution.        E56. The method of E54 or E55, wherein the at least two        different solutions (e.g., a first gradient elution buffer, a        second gradient elution buffer) comprise a component selected        from the group consisting of a buffering agent, a salt, a        detergent, and a combination thereof.        E57. The method of E56, wherein the buffering agent is selected        from the group consisting of Tris, BIS-Tris propane,        diethanolamine, diethylamine, tricine, triethanolamine and/or        bicine.        E58. The method of E56 or E57, wherein the salt is selected from        the group consisting of sodium chloride, sodium acetate,        ammonium acetate, magnesium chloride, sodium sulfate and a        combination thereof.        E59. The method of any one of E56-E58, wherein the detergent is        selected from the group consisting of poloxamer 188 (P188),        Triton X-100, polysorbate 80 (PS80), Brij-35, nonyl        phenoxypolyethoxylethanol (NP-40) and a combination thereof.        E60. The method of any one of E55-E59, wherein the at least two        different solutions have different pH, salt concentration,        conductivity and/or modifier concentration.        E61. The method of any one of E55-E60, wherein the first        solution (e.g., buffer A) comprises about 50 mM to about 150 mM        (e.g., about 100 mM) Tris, about 0.005% to about 0.015% (e.g.,        about 0.01%) P188, about pH 8.5 to 9.5 (e.g., about 8.9).        E62. The method of any one of E55-E61, wherein the second        solution (e.g., buffer B) comprises about 400 mM to about 600 mM        (e.g., about 500 mM) sodium acetate, about 50 mM to about 150 mM        (e.g., about 100 mM) Tris, about 0.005% to about 0.015% (e.g.,        about 0.01%) P188, about pH 8.5 to 9.5 (e.g., about 8.9).        E63. The method of any one of E55-E62, wherein performing the        gradient elution comprises application of about 10 to 60 CV,        about 20 to 40 CV or about 20 to 24 CV of the at least two        different solutions to the stationary phase.        E64. The method of any one of E55-E63, wherein at the start of        the gradient elution the percentage of the first solution (e.g.,        a first gradient elution buffer, buffer A) is 50% to 100% and at        the end of the gradient elution the percentage of the second        solution (e.g., second gradient elution buffer, buffer B) is 50%        to 100%, and wherein optionally 10 to 60 CV of the first        solution, the second solution or a mixture of both are applied        to the stationary phase during the gradient elution.        E65. The method of any one of E55-E64, wherein at the start of        the gradient elution the percentage of the first solution (e.g.,        a first gradient elution buffer, buffer A) is 100% and at the        end of the gradient elution the percentage of the second        solution (e.g., second gradient elution buffer, buffer B) is        100%, and wherein optionally 10 to 60 CV of the first solution,        the second solution or a mixture of both are applied to the        stationary phase during the gradient elution.        E66. The method of any one of E55-E65, wherein a concentration        of a component of the first solution (e.g., first gradient        elution buffer) or second solution (e.g., second gradient        elution buffer) increases or decreases continuously during the        gradient elution; wherein a rate of increase or decrease of a        concentration of a component of the first solution or second        solution is equivalent to a change in concentration of a        component per total CV; and wherein the rate of change in        concentration of a component over a gradient elution is about 10        mM/CV to 50 mM/CV.        E67. The method of any one of E54-E66, wherein full capsids are        eluted from the stationary phase in a first elution peak and/or        in a first portion of a second elution peak of the gradient        elution.        E68. The method of any one of E54-E67, wherein empty capsids are        recovered in an AEX column flow-through and/or in a last portion        of a second elution peak of the gradient elution.        E69. The method of any one of E1-E68, comprising performing a        gradient hold.        E70. The method of E69, wherein the gradient hold comprises        application of 1 to 10 CV of a gradient hold solution comprising        a component selected from the group consisting of a salt, a        buffering agent, a detergent and a combination thereof to the        stationary phase in the column.        E71. The method of E69 or E70, wherein performing the gradient        hold comprises application of 1 to 10 CV of a gradient hold        solution comprising about 5 mM to about 1 M (e.g., about 500 mM)        sodium acetate, 50 mM to 150 mM (e.g., about 100 mM) Tris, about        0.005% to about 0.015% (e.g., about 0.01%) P188, pH about 8.5 to        9.5 (e.g., about 8.9) to the AEX stationary phase in the column.        E72. The method of any one of E1-E71, comprising performing a        step elution (e.g., isocratic elution) from the stationary phase        in the column.        E73. The method of E72, wherein performing the step elution        comprises application of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or        more step elution solutions to the column stationary phase.        E74. The method of E72 or E73, wherein the at least 2, 3, 4, 5,        6, 7, 8, 9, 10 or more step elution solutions each comprise a        component selected from the group consisting of a buffering        agent, a salt, a detergent and a combination thereof.        E75. The method of any one of E72-E74, wherein the at least 2,        3, 4, 5, 6, 7, 8, 9, 10 or more step elution solutions each        comprise about 10 mM to about 50 mM (e.g., about 20 mM) Tris and        about 5 mM to about 600 mM sodium acetate, pH about 8 to 10        (e.g., about 8.9 to about 9.1).        E76. The method of any one of E72-E75, wherein step elution        solutions with increasing concentration of sodium acetate are        sequentially applied to the column.        E77. The method of any one of E72-E76, wherein the final step        elution solution comprises about 20 mM Tris, about 500 mM sodium        acetate, pH about 8.9 to about 9.1.        E78. The method of any one of E1-E77, comprising collecting at        least one fraction of eluate from the column to recover full        rAAV capsids, optionally during a gradient elution.        E79. The method of E78, wherein a volume of the at least one        fraction of eluate is selected from the group consisting of ⅛ of        a CV, ¼ of a CV, ⅓ of a CV, ½ of a CV, ¾ of a CV, 1 CV, 2 CV, 3        CV, 4 CV, 5 CV, 6 CV, 7 CV, 8 CV, 9 CV, 10 CV or more.        E80. The method of E79, wherein the absorbance of the at least        one fraction of eluate is measured at 280 nm, and wherein        optionally, the threshold is ≥0.5 mAU/mm path length measured at        280 nm.        E81. The method of E80, wherein the at least one fraction of        eluate is collected when the A280 of the eluate is ≥0.5 mAU/mm        path length.        E82. The method of E80 or E81, wherein a volume of the at least        one fraction of eluate is equivalent to ⅛ of a CV to 10 CV,        e.g., ⅛ of a CV, ¼ of a CV, ⅓ of a CV, ½ of a CV, ¾ of a CV, 1        CV, 2 CV, 3 CV, 4 CV, 5 CV, 6 CV, 7 CV, 8 CV, 9 CV, 10 CV or        more of a CV, and wherein optionally, the A260/A280 ratio of the        at least one fraction of eluate is ≥ to 1.25.        E83. The method of any one of E78-E82, wherein at least 2, at        least 3, at least 4, at least 5, at least 6, at least 7, at        least 8, at least 9, at least 10, at least 11, at least 12, at        least 13, at least 14, at least 15, at least 16, at least 17, at        least 18, at least 19, at least 20, at least 21, at least 22, at        least 23, at least 24, at least 25, or more fractions of eluate        are collected.        E84. The method of any one of E78-E83, further comprising        adjusting a pH of the at least one fraction of eluate collected        from the column, optionally during a gradient elution.        E85. The method of E84, wherein adjusting the pH of the at least        one fraction of eluate comprises i) addition of 14.3% to 15%        eluate volume by weight of a solution comprising a buffering        agent, pH about 3.5 to the at least one fraction of eluate        or ii) collecting the at least one fraction of eluate into a        vessel comprising about 0.01 CV to 0.1 CV (e.g., about 0.066 CV)        of a solution comprising a buffering agent.        E86. The method of E85, wherein the buffering agent is about 200        mM to about 300 mM (e.g., about 250 mM) sodium citrate.        E87. The method of E84-E86, wherein the pH of the at least one        fraction of eluate collected from the column is adjusted from an        initial pH of about 8.5 to about 9.1 to a pH of about 6.8 to        about 7.6.        E88. The method of E84-E87, wherein the pH of the at least one        fraction of eluate collected from the column is adjusted from an        initial pH of about 8.5 to about 9.1 to a pH of about 7.0 to        about 7.4.        E89. The method of any one of E78-E88, further comprising        measuring an absorbance of at least one fraction of eluate        collected from the column, optionally during a gradient elution.        E90. The method of E89, wherein the absorbance is measured at        260 nm (A260), 280 nm (A280) or at 260 nm and 280 nm, and        optionally wherein an A260/A280 ratio is determined.        E91. The method of E90, wherein the absorbance at 260 nm and 280        nm is measured by size exclusion chromatography (SEC).        E92. The method of E90 or E91, wherein the A260/A280 ratio of at        least one fraction of eluate is at least 0.5, at least 0.55, at        least 0.6, at least 0.65, at least 0.70, at least 0.75, at least        0.80, at least 0.85, at least 0.90, at least 0.95. at least 1.0,        at least, 1.05, least 1.10, at least 1.11, at least 1.12, at        least 1.13, at least 1.14, at least 1.15, at least 1.16, at        least 1.17, at least 1.18, at least 1.19, at least 1.20, at        least 1.21, at least 1.22, at least 1.23, at least 1.24, at        least 1.25, at least 1.26, at least 1.27, at least 1.28, at        least 1.29, at least 1.30, at least 1.31, at least 1.32, at        least 1.33, at least 1.34, at least 1.35, at least 1.36, at        least 1.37, at least, 1.38, at least 1.39, at least 1.40 or is        about 0.5 to about 2.0, about 0.5 to about 1.8, about 0.5 to        about 1.6, about 0.5 to about 1.4, about 0.5 to about 1.2, about        0.5 to about 1.0, about 0.5 to about 0.8, about 0.6 to about        2.0, about 0.8 to about 1.8, about 0.8 to about 1.6, about 0.8        to about 1.4, about 1.0 to about 1.4, or about 1.0 to about 1.2,        optionally as measured by SEC.        E93. The method of any one of E90 to E92, wherein the A260/A280        ratio of at least one fraction of eluate is at least 1.25.        E94. The method of any one of E78-E93, further comprising        combining at least two fractions of eluate collected from the        column, optionally during a gradient elution to form a pooled        eluate comprising the rAAV vector.        E95. The method of E94, wherein 2 to 50 fractions of eluate are        combined to form a pooled eluate.        E96. The method of E94 or E95, wherein the at least two        fractions of eluate each have an A260/A280 ratio of ≥1.25.        E97. The method of any one of E94-E96, further comprising        measuring the absorbance of the pooled eluate, and wherein the        A260/A280 of the pooled eluate is ≥1.25 (e.g., about 1.28 to        1.35).        E98. The method of any one of E94-E97, wherein the pooled eluate        has a pH of 6.8 to 7.6 (e.g., 7.0 to 7.4).        E99. The method of any one of E78-E98, wherein full capsids        comprise 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to        95%, 20% to 98%, 20% to 99%, 20% to greater than 99%, 40% to        50%, 40% to 60%, 40% to 70%, 40% to 80% (e.g., 44%, 45%, 50%,        53%) of total capsids in the at least one fraction of eluate, or        the pooled eluate, and optionally wherein the capsids are        measured by analytical ultracentrifugation (AUC).        E100. The method of any one of E78-E99, wherein full capsids        comprise about 55% of total capsids (e.g., 55%+/−7%) in the at        least one fraction of eluate, or the pooled eluate.        E101. The method of any one of E78-E99, wherein full capsids        comprise about 49% (e.g., 49%+/−2%) of total capsids in the at        least one fraction of eluate, or the pooled eluate.        E102. The method of any one of E78-E99, wherein full capsids        comprise 52+/−7% of total capsids in the at least one fraction        of eluate, or the pooled eluate.        E103. The method of any one of E78-E99, wherein the at least one        fraction of eluate, or the pooled eluate, comprises 48% to 62%        full rAAV capsids of total capsids, and wherein the at least one        fraction of eluate, or the pooled eluate, is generated from        purification of a rAAV vector produced in a 250 L SUB.        E104. The method of any one of E78-E99, wherein the at least one        fraction of eluate, or the pooled eluate, comprises 47% to 51%        full rAAV capsids, of total capsids, and wherein the at least        one fraction of eluate, or the pooled eluate, is generated from        purification of a rAAV vector produced in a 2000 L SUB.        E105. The method of any one of E78-E99, wherein the at least one        fraction of eluate, or the pooled eluate, comprises greater than        30% (e.g., 40% to 55%, 45% to 65%, 40% to greater than 99%) full        capsids of total capsids and wherein the solution comprising the        rAAV vector to be purified comprises less than 30% (e.g., 12% to        25%) full capsids of total capsids in the solution.        E106. The method of any one of E78-E105, wherein empty capsids        comprise 10% to 99%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to        65%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%,        20% to 65%, 20% to 60%, 20% to 50%, 20% to 40%, or 18% to 29%,        (e.g., ≤29%) of total capsids in the at least one fraction of        eluate, or the pooled eluate, and optionally wherein the capsids        are measured by analytical ultracentrifugation (AUC).        E107. The method of any one of E78-E106, wherein empty capsids        comprise 20%+/−7% (e.g., 21%) of total capsids in the at least        one fraction of eluate, or the pooled eluate.        E108. The method of any one of E78-E106, wherein the at least        one fraction of eluate, or the pooled eluate, comprises 11% to        31% empty capsids of total capsids, and wherein the at least one        fraction of eluate, or the pooled eluate, is generated from        purification of a rAAV vector produced in a 250 L SUB.        E109. The method of any one of E78-E106, wherein the at least        one fraction of eluate, or the pooled eluate, comprises 18% to        22% empty capsids of total capsids, and wherein the at least one        fraction of eluate, or the pooled eluate, is generated from        purification of a rAAV vector produced in a 2000 L SUB.        E110. The method of any one of E78-E106, wherein the at least        one fraction of eluate or the pooled eluate comprises less than        30% empty capsids of the total capsids, and wherein the solution        comprising the rAAV vector to be purified comprises 40% to 90%        empty capsids of total capsids in the solution.        E111. The method of any one of E78-E110, wherein intermediate        capsids comprise 10% to 65%, 10% to 60%, 10% to 50%, 10% to 40%,        10% to 30%, 10% to 20%, 20% to 65%, 20% to 60%, 20% to 50%, 20%        to 40%, or 18% to 22% of total capsids in the at least one        fraction of eluate, or the pooled eluate, and optionally wherein        the capsids are measured by analytical ultracentrifugation        (AUC).        E112. The method of any one of E78-E111, wherein intermediate        capsids comprise 28%+/−5% of total capsids in the at least one        fraction of eluate, or the pooled eluate.        E113. The method of any one of E78-E111, wherein the at least        one fraction of eluate, or the pooled eluate, comprises 21% to        27% intermediate capsids of total capsids, and wherein the at        least one fraction of eluate or the pooled eluate is generated        from purification of a rAAV vector produced in a 250 L SUB.        E114. The method of any one of E78-E111, wherein the at least        one fraction of eluate, or the pooled eluate, comprises 28% to        36% intermediate capsids of total capsids, and wherein the at        least one fraction of eluate, or the pooled eluate, is generated        from purification of a rAAV vector produced in a 2000 L SUB.        E115. The method of any one of E78-E111, wherein the at least        one fraction of eluate, or the pooled eluate, comprises 45% to        65% full rAAV capsids, 19% to 28% intermediate capsids and 10%        to 37% empty capsids of total capsids, and wherein the at least        one fraction of eluate or the pooled eluate is generated from        purification of a rAAV vector produced in a 250 L SUB.        E116. The method of E115, wherein full capsids comprise 55%+/−7%        of total capsids in the at least one fraction of eluate or the        pooled eluate.        E117. The method of E115, wherein intermediate capsids comprise        24%+/−3% of total capsids in the at least one fraction of eluate        or the pooled eluate.        E118. The method of E115, wherein empty capsids comprise        21%+/−10% of total capsids in the at least one fraction of        eluate or the pooled eluate.        E119. The method of any one of E78-E118, wherein the at least        one fraction of eluate, or the pooled eluate, comprises 45% to        52% full rAAV capsids, 27% to 37% intermediate capsids and/or        18% to 22% empty capsids of total capsids, and wherein the at        least one fraction of eluate, or the pooled eluate, is generated        from purification of a rAAV vector produced in a 2000 L SUB.        E120. The method of E119, wherein full capsids comprise 49%+/−2%        of total capsids in the at least one fraction of eluate or the        pooled eluate.        E121. The method of E119, wherein the intermediate capsids        comprise 32%+1-4% of total capsids in the at least one fraction        of eluate or the pooled eluate.        E122. The method of E119, wherein the empty capsids comprise        20%+/−2% of total capsids in the at least one fraction of eluate        or the pooled eluate.        E123. The method of any one of E94-E122, wherein the pooled        eluate is enriched for full capsids, and/or depleted of empty        capsids, as compared to the solution loaded onto the column.        E124. The method of any one of E78-E123, wherein a % VG step        yield of the at least one fraction of eluate, or the pooled        eluate, is 1% to 10%, 1 to 20%, 1% to 30%, 1% to 40%, 1% to 50%,        1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 99%, 5% to        95%, 10% to 85%, 15% to 75%, 20% to 65%, 25% to 55%, 30% to 45%,        30% to 80%, 40% to 70% or 100%.        E125. The method of E124, wherein the % VG step yield of the at        least one fraction of eluate, or the pooled eluate, is 31% to        66% (e.g., 47%+/−11%).        E126. The method of E124, wherein the % VG step yield of an at        least one fraction of eluate or a pooled eluate produced in a        250 L SUB is 30% to 70% (e.g., 37% to 60%).        E127. The method of E124, wherein the % VG step yield of the at        least one fraction of eluate or the pooled eluate produced in a        250 L SUB is 45%+/−8%.        E128. The method of E124, wherein the % VG step yield of an at        least one fraction of eluate or a pooled eluate produced in a        2000 L SUB is 25% to 75% (e.g., 31% to 66%).        E129. The method of E124, wherein the % VG step yield of the at        least one fraction of eluate or the pooled eluate produced in a        2000 L SUB is 50%+/−13%.        E130. The method of any one of E124-E129, wherein the % VG step        yield of the at least one fraction of eluate, or the pooled        eluate, is greater than the % VG step yield of an otherwise        identical fraction of eluate, or pooled eluate, purified by        ultracentrifugation and cation exchange chromatography.        E131. The method of any one of E78-E130, wherein the A260/A280        (SEC) of the at least one fraction of eluate, or the pooled        eluate, produced in a 250 L SUB is 1.29+/−0.03.        E132. The method of any one of E78-E130, wherein the A260/A280        (SEC) of the at least one fraction of eluate, or the pooled        eluate, produced in a 2000 L SUB is 1.30+/−0.01        E133. The method of any one of E78-E132, wherein a % VG column        yield of the at least one fraction of eluate, or the pooled        eluate, is 1% to 10%, 1 to 20%, 1% to 30%, 1% to 40%, 1% to 50%,        1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 99%, 5% to        95%, 10% to 85%, 15% to 75%, 20% to 65%, 25% to 55%, 30% to 45%,        30% to 80%, 40% to 70% or 100%.        E134. The method of E133, wherein the % VG column yield of the        at least one fraction of eluate, or the pooled eluate, is 20% to        100% (e.g., 63%+/−26%).        E135. The method of E133, wherein the % VG column yield of an at        least one fraction of eluate, or a pooled eluate, produced in a        250 L SUB is 40% to 100%.        E136. The method of E133, wherein the % VG column yield of an at        least one fraction of eluate, or a pooled eluate, produced in a        2000 L SUB is 10% to 70% (e.g., 20% to 61%).        E137. The method of any one of E78-E136, wherein the at least        one fraction of eluate, or pooled eluate, is further subjected        to a method of filtration selected from the group consisting of        viral filtration, ultrafiltration/diafiltration (UF/DF),        filtration through a 0.2 μm filter and a combination thereof, to        produce a drug substance.        E138. The method of E137, wherein full capsids comprise 20% to        60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 98%,        20% to 99%, 20% to greater than 99%, 40% to 50%, 40% to 60%, 40%        to 70%, 40% to 80% (e.g., 44%, 45%, 50%, 53%) of total capsids        in the drug substance, and optionally wherein the capsids are        measured by analytical ultracentrifugation (AUC).        E139. The method of E137 or E138, wherein the drug substance        comprises 45% to 65% full rAAV capsids, of total capsids, and        optionally wherein the drug substance is generated from        purification of a rAAV vector produced in a 250 L SUB.        E140. The method of any one of E137-E139, wherein full capsids        comprise 52+/−7% of total capsids in the drug substance        E141. The method of E137 or E138, wherein the drug substance        comprises 45% to 52% full rAAV capsids, of total capsids, and        wherein the drug substance is generated from purification of a        rAAV vector produced in a 2000 L SUB.        E142. The method of E137 or E138, wherein the drug substance        comprises greater than 30% (e.g., 40% to 55%, 45% to 65%, 40% to        greater than 99%) full capsids of total capsids in the drug        substance, and wherein the solution comprising the rAAV vector        to be purified comprises less than 30% (e.g., 12% to 25%) full        capsids of total capsids in the solution.        E143. The method of any one of E137-E142, wherein empty capsids        comprise 10% to 99%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to        65%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%,        20% to 65%, 20% to 60%, 20% to 50%, 20% to 40%, or 18% to 29%,        (e.g., ≤29%) of total capsids in the drug substance, and        optionally wherein the capsids are measured by analytical        ultracentrifugation (AUC).        E144. The method of any one of E143, wherein the drug substance        comprises 10% to 37% empty capsids of total capsids, and        optionally wherein the drug substance is generated from        purification of a rAAV vector produced in a 250 L SUB.        E145. The method of E143 or E144, wherein empty capsids comprise        20%+/−7% of total capsids in the drug substance.        E146. The method of any one of E143, wherein the drug substance        comprises 18% to 22% empty capsids of total capsids, and        optionally wherein the drug substance is generated from        purification of a rAAV vector produced in a 2000 L SUB.        E147. The method of any one of E143-E146, wherein the drug        substance comprises less than 30% empty capsids of the total        capsids in the drug substance, and wherein the solution        comprising the rAAV vector to be purified comprises 40% to 90%        empty capsids of total capsids in the solution.        E148. The method of any one of E137-E147, wherein intermediate        capsids comprise 10% to 65%, 10% to 60%, 10% to 50%, 10% to 40%,        10% to 30%, 10% to 20%, 20% to 65%, 20% to 60%, 20% to 50%, 20%        to 40%, or 18% to 22% of total capsids in the drug substance,        and optionally wherein the capsids are measured by analytical        ultracentrifugation (AUC).        E149. The method of any one of E137-E148, wherein the drug        substance comprises 19% to 37% intermediate capsids of total        capsids, and optionally wherein the drug substance is generated        from purification of a rAAV vector produced in a 250 L or 2000 L        SUB.        E150. The method of E148 or E149, wherein intermediate capsids        comprise 28%+/−5% of total capsids in the drug substance.        E151. The method of any one of E137-E150, wherein the drug        substance comprises 45% to 65% full rAAV capsids, 19% to 28%        intermediate capsids and 10% to 37% empty capsids of total        capsids, and wherein the drug substance is generated from        purification of a rAAV vector produced in a 250 L SUB.        E152. The method of E151, wherein the full capsids comprise        55%+/−7% of total capsids.        E153. The method of E151 or E152, wherein the intermediate        capsids comprise 24%+/−3% of total capsids.        E154. The method of any one of E151-E153, wherein the empty        capsids comprise 21%+/−10% of total capsids.        E155. The method of any one of E137-E150, wherein the drug        substance comprises 45% to 52% full rAAV capsids, 27% to 37%        intermediate capsids and/or 18% to 22% empty capsids of total        capsids, and wherein the drug substance is generated from        purification of a rAAV vector produced in a 2000 L SUB.        E156. The method of E155, wherein the full capsids comprise        49%+/−2% of total capsids.        E157. The method of E155 or E156, wherein the intermediate        capsids comprise 32%+/−4% of total capsids.        E158. The method of any one of E155-E157, wherein the empty        capsids comprise 20%+/−2% of total capsids.        E159. The method of any one of E137-E158, wherein the drug        substance is depleted of host cell protein (HCP) as compared to        the amount of HCP in the solution comprising the rAAV vector to        be purified.        E160. The method of any one of E137-E159, wherein the drug        substance comprises an amount of HCP below a lowest level of        quantification (LLOQ) as measured by ELISA.        E161. The method of any one of E137-E160, wherein the drug        substance comprises an amount of HCP that is below the LLOQ, as        measured by ELISA, wherein the solution comprising the rAAV        vector to be purified comprises about 1 to 500 pg HCP/1×10⁹ VG,        and optionally wherein the solution is generated from affinity        chromatography purification of a rAAV vector produced in a 250 L        SUB.        E162. The method of any one of E137-E161, wherein the drug        substance comprises an amount of HCP that is below the LLOQ, as        measured by ELISA, wherein the solution comprising the rAAV        vector to be purified comprises about 100 to 1000 pg HCP/1×10⁹        VG, and optionally wherein the solution is generated from        affinity chromatography purification of a rAAV vector produced        in a 2000 L SUB.        E163. The method of any one of E137-E162, wherein the purity of        the drug substance is about 90%, about 91%, about 92%, about        93%, about 94%, about 95%, about 96%, about 97%, about 98%,        about 99% or 100%, optionally, as measured by reverse phase        HPLC.        E164. The method of E163, wherein the purity of the drug        substance is about 98.6+/−0.6% and optionally wherein the drug        substance is generated from purification of a rAAV vector        produced in a 250 L SUB.        E165. The method of E164, wherein the purity of the drug        substance is about 99.3+/−0.3% and optionally wherein the drug        substance is generated from purification of a rAAV vector        produced in a 2000 L SUB.        E166. The method of any one of E137-E165, wherein the drug        substances comprises about 0 to 10% HMMS, optionally, as        measured by SEC.        E167. The method of E166, wherein the drug substance comprises        2.6+/−0.8% HMMS and optionally wherein the drug substance is        generated from purification of a rAAV vector produced in a 250 L        SUB.        E168. The method of E166, wherein the drug substance comprises        2.9+/−0.4% HMMS and optionally wherein the drug substance is        generated from purification of a rAAV vector produced in a 2000        L SUB.        E169. The method of E137-E168, wherein the drug substance        comprises about 7.0 to 25 pg/1×10⁹ VG of residual HC-DNA.        E170. The method of E169, wherein the drug substance comprises        about 17.4+/−6.7 pg/1×10⁹ VG of HC-DNA and optionally wherein        the drug substance is generated from purification of a rAAV        vector produced in a 250 L SUB.        E171. The method of E169, wherein the drug substance comprises        about 9.3+/−1.2 pg/1×10⁹ VG of HC-DNA, and optionally wherein        the drug substance is generated from purification of a rAAV        vector produced in a 2000 L SUB.        E172. The method of any one of E137-E171, wherein the drug        substance has an A260/A280 of about 1.24 to 1.32, optionally, as        measured by size exclusion chromatography (SEC).        E173. The method of E172, wherein the drug substance has an        A260/A280 of 1.24 to 1.32, optionally, as measured by SEC, and        wherein the drug substance is generated from purification of a        rAAV vector produced in a 250 L SUB.        E174. The method of E172, wherein the drug substance has an        A260/A280 of 1.28 to 1.31, optionally, as measured by SEC, and        optionally wherein the drug substance is generated from        purification of a rAAV vector produced in a 2000 L SUB.        E175. The method of any one of E1-E174, wherein a volume of the        column is 1.0 mL to 6.6 L.        E176. The method of any one of E1-E175, wherein the volume of        the column is about 1.0 mL, about 5.1 mL, about 6.67 mL, about        1.256 L, about 1.3 L, about 6.3 L, about 6.4 L, or about 6.6 L.        E177. The method of any one of E1-E176, wherein the rAAV vector        comprises a capsid protein from an AAV serotype selected from        the group consisting of AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4,        AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAVrh74, AAV12,        AAV2i8, NP4, NP22, NP66, AAVDJ, AAVDJ/8, AAVDJ/9, AAVLK03,        AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO:5        of WO 2015/013313), RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4,        RHM15-6, AAV hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45,        AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N,        AAV3B-S312N, AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5,        AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11,        AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15.        E178. The method of any one of E1-E177, wherein a purified rAAV        vector is produced.        E179. The method of E178, wherein the purified rAAV vector is a        drug substance.        E180. The method of any one of E137-E174 or E179, wherein the        drug substance and a pharmaceutically acceptable excipient are        combined to form a drug product.        E181. The method of any one of E178-E180, wherein the purified        rAAV vector, the drug substance and/or the drug product is        suitable for administration to a subject to treat a disease,        disorder or condition.        E182. The method of E181, wherein the disease, disorder or        condition is Duchenne muscular dystrophy (DMD).        E183. The method of any one of E1-E182, wherein the rAAV vector        comprises a vector genome comprising a modified nucleic acid        encoding a human mini-dystrophin.        E184. The method of E183, wherein the modified nucleic acid        comprises or consists of the nucleic acid sequence of SEQ ID NO:        1.        E185. The method of E183 or E184, wherein the modified nucleic        acid encodes a human mini-dystrophin comprising or consisting of        the amino acid sequence of SEQ ID NO:2.        E186. The method of any one of E183-E185, wherein the vector        genome comprises a muscle specific promoter and/or enhancer        selected from the group consisting of a synthetic hybrid        muscle-specific promoter hCK, a synthetic hybrid muscle-specific        promoter hCKplus, and a synthetic muscle-specific enhancer and        promoter.        E187. The method of E186, wherein the synthetic hybrid        muscle-specific promoter hCK comprises or consists of the        nucleic acid sequence of SEQ ID NO:3.        E188. The method of E186, wherein the synthetic hybrid        muscle-specific promoter hCKplus comprises or consists of the        nucleic acid sequence of SEQ ID NO:4.        E189. The method of E186, wherein the synthetic muscle-specific        enhancer and promoter comprises or consists of the nucleic acid        sequence of SEQ ID NO:5.        E190. The method of any one of E183-E189, wherein the vector        genome comprises a polyadenylation (polyA) signal sequence.        E191. The method of E190, wherein the polyA signal sequence        comprises or consists of the nucleic acid sequence of SEQ ID        NO:6.        E192. The method of any one of E183-E191, wherein the vector        genome comprises a transcription terminator sequence.        E193. The method of E192, wherein the transcription terminator        sequence comprises or consists of the nucleic acid sequence of        SEQ ID NO:9.        E194. The method of any one of E183-E193, wherein the vector        genome comprises at least one ITR.        E195. The method of E194, wherein the at least one ITR comprises        or consists of a nucleic acid sequence selected from SEQ ID        NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14 and a combination        thereof.        E196. The method of any one of E183-E195, wherein the vector        genome comprises an expression cassette, and wherein the        expression cassette comprises or consists of the nucleic acid        sequence of SEQ ID NO:10.        E197. The method of any one of E1-E196, wherein the rAAV vector        comprises a VP1 polypeptide of AAV9.        E198. The method of E197, wherein the VP1 polypeptide comprises        or consists of the amino acid sequence of SEQ ID NO:11.        E199. The method of any one of E1-E198, wherein the rAAV vector        comprises an AAV9 capsid protein and a transgene comprising the        nucleic acid sequence of SEQ ID NO:1.        E200. The method of any one of E1-E199, further comprising        preparing the solution comprising the rAAV vector for        purification by AEX.        E201. The method of E200, wherein the solution comprising the        rAAV vector is selected from the group consisting of an affinity        eluate, a supernatant from a cell lysate and a post-harvest        solution that has been diluted and optionally filtered prior to        loading.        E202. The method of E200 or E201, wherein the solution        comprising the rAAV vector is an affinity eluate that has been        diluted and optionally filtered prior to loading.        E203. The method of any one of E200-E202, wherein the solution        has already undergone at least one purification and/or        processing step.        E204. The method of any one of E200-E203, wherein the solution        comprising the rAAV vector is an eluate resulting from affinity        chromatography purification of a rAAV vector.        E205. The method of any one of E200-E204, wherein preparation        comprises diluting the solution comprising the rAAV vector        E206. The method of E205, wherein diluting the solution        comprising the rAAV vector comprises diluting the solution about        2-fold, about 3-fold, about 4-fold, about about, 6-fold, about        7-fold, about 8-fold, about 9-fold, about 10-fold, about        11-fold, about 12-fold, about 13-fold, about 14-fold, about        15-fold, about 16-fold, about 17-fold, about 18-fold, about-19        fold, about 20-fold, about 25-fold to produce a diluted        solution.        E207. The method of E205 or E206, wherein diluting the solution        comprising the rAAV vector comprises diluting the solution about        15-fold to produce a diluted solution.        E208. The method of any one of E205-E207, wherein diluting the        solution comprising the rAAV vector is performed in-line with        the AEX column, and wherein a dilution solution is delivered        through a first tubing to a Y-connector, and the solution        comprising the rAAV vector is delivered through a second tubing        to the Y-connector.        E209. The method of E208, wherein the dilution solution is        delivered at a flow rate of 1 to 5 mL/min, and wherein the        solution comprising the rAAV vector is delivered at a flow rate        of 0.1 to 2 mL/min, such that the solution is diluted about        15-fold.        E210. The method of E208 or E209, wherein the dilution solution        comprises at least one component selected from the group        consisting of a buffering agent, an amino acid, a detergent and        a combination thereof.        E211. The method of E210, wherein the buffering agent is        selected from the group consisting of Tris, BIS-Tris propane,        diethanolamine, diethylamine, tricine, triethanolamine, bicine        and a combination thereof.        E212. The method of E210 or E211, wherein the amino acid is        selected from the group consisting of histidine, arginine,        glycine, citrulline and a combination thereof.        E213. The method of any one of E210-E212, wherein the amino acid        is histidine.        E214. The method of any one of E210-E213, wherein the detergent        is selected from the group consisting of poloxamer 188 (P188),        Triton X-100, polysorbate 80 (PS80), Brij-35, nonyl        phenoxypolyethoxylethanol (NP-40) and a combination thereof.        E215. The method of any one of E210-E214, wherein the detergent        is P188.        E216. The method of any one of E208-E215, wherein the dilution        solution comprises about 100 mM to about 300 mM (e.g., about 200        mM) histidine, about 100 mM to about 300 mM (e.g., about 200 mM)        Tris, about 0.1% to about 1.0% (e.g., about 0.5%) P188, pH about        8.5 to about 9.5 (e.g., about 8.8).        E217. The method of any one of E205-E216, wherein diluting the        solution comprising the rAAV vector precedes loading the        solution comprising the rAAV vector onto the column.        E218. The method of any one of E205-E217, wherein pH of the        solution comprising the rAAV vector after diluting is increased        as compared to the pH of the solution before diluting.        E219. The method of any one of E205-E218, wherein pH of the        solution comprising the rAAV vector before diluting is 3.0 to        4.4 and the pH of the solution after diluting is 8.5 to 9.5.        E220. The method of any one of E205-E219, wherein conductivity        of the solution comprising the rAAV vector diluting is decreased        as compared to conductivity of the solution before diluting.        E221. The method of any one of E205-E220, wherein conductivity        of the solution comprising the rAAV vector before diluting is        5.0 to 7.0 mS/cm (e.g., 5.5 to 6.5 mS/cm) and conductivity of        the solution after diluting is 1.7 to 3.5 mS/cm.        E222. The method of any one of E205-E221, further comprising        filtering the diluted solution comprising the rAAV vector.        E223. The method of E222, wherein filtering the diluted solution        comprises filtration through a 0.2 μm filter.        E224. The method of E222 or E223, wherein the filter is in-line        with the column.        E225. The method of any one of E222-E224, wherein diluting and        filtering the solution comprising the rAAV vector precedes        loading the solution comprising the rAAV vector onto the column.        E226. The method of any one of E205-E225, wherein the percent        vector genome (% VG) yield of the diluted, and optionally        filtered solution comprising the rAAV vector is 60% to 100% as        compared to the amount of VG present in the solution prior to        dilution, and optionally filtration.        E227. The method of any one of E205-E226, wherein a % VG        dilution yield of the diluted solution comprising the rAAV        vector is 88%+/−36%.        E228. A method of preparing a solution comprising a rAAV vector        for purification by AEX, the method comprising a step of:    -   i) diluting a first solution 2 to 25-fold (e.g., 15-fold) with a        dilution solution; and optionally    -   ii) filtering the solution from step i) through a filter to        produce the diluted, and optionally filtered solution; wherein        the pH of the diluted, and optionally filtered solution is        increased as compared to the pH of the first solution; and        wherein the conductivity of the diluted, and optionally filtered        solution is decreased as compared to the conductivity of the        first solution.        E229. The method of preparing a solution comprising a rAAV        vector for purification by AEX of E228, wherein i) the pH of the        diluted, and optionally filtered solution is 8.5 to 9.5; ii) the        conductivity of the diluted, and optionally filtered solution is        1.7 to 3.3 mS/cm; and/or iii) a % VG dilution yield of the        solution after dilution is 35% to 100%.        E230. The method of preparing a solution comprising a rAAV        vector for purification by AEX of E228 or E229, wherein the rAAV        vector comprises an AAV9 capsid protein.        E231. A method of purifying an rAAV vector by AEX, the method        comprising a step of:    -   i) loading a solution comprising the rAAV vector to be purified        onto an AEX stationary phase in a column;    -   ii) performing gradient elution of a material from the        stationary phase in the column wherein a percentage of a first        gradient elution buffer is varied in a manner inversely        proportional to variation in a percentage of a second gradient        elution buffer;    -   iii) collecting at least one fraction of eluate from the column        during the gradient elution beginning when the absorbance of a        column flow through reaches an absorbance threshold;    -   iv) measuring an absorbance of the at least one fraction of        eluate collected from the column and determining an A260/A280        ratio.        E232. A method of purifying an rAAV vector by AEX of E231,        wherein the method further comprises combining at least two        fractions of eluate collected from the column to form a pooled        eluate comprising the rAAV vector.        E233. The method of purifying a rAAV vector by AEX of E231 or        E232, wherein the AEX stationary phase is POROS™ 50 HQ.        E234. The method of purifying a rAAV vector by AEX of any one of        E231-E233, wherein the solution is an affinity eluate has been        diluted and filtered prior to loading onto the stationary phase.        E235. The method of purifying a rAAV vector of any one of        E231-E234, wherein the material eluted from the stationary phase        comprises the rAAV vector to be purified.        E236. The method of purifying a rAAV vector by AEX of any one of        E231-E235, wherein a first gradient elution buffer comprises        about 50 mM to about 150 mM (e.g., about 100 mM) Tris, about        0.005% to about 0.015% (e.g., about 0.01%) P188, about pH 8.5 to        9.5 (e.g., about 8.9).        E237. The method of purifying a rAAV vector by AEX of any one of        E231-E236, wherein a second gradient elution buffer comprises        about 400 mM to about 600 mM (e.g., about 500 mM) sodium        acetate, about 50 mM to about 150 mM (e.g., about 100 mM) Tris,        about 0.005% to about 0.015% (e.g., about 0.01%) P188, about pH        8.5 to 9.5 (e.g., about 8.9).        E238. The method of purifying a rAAV vector by AEX of any one of        E231-E237 wherein at the start of the gradient elution the        percentage of the first gradient elution buffer is 100% and at        the end of the gradient elution the percentage of the second        gradient elution buffer is 100%.        E239. The method of purifying a rAAV vector by AEX of any one of        E236-E238, wherein about 15 to about 25 (e.g., 20 CV) of the        first gradient elution buffer, the second gradient elution        buffer or a mixture of both are applied to the stationary phase        during the gradient elution; wherein a concentration of the        sodium acetate varies from 0 mM to 500 mM during the gradient        elution such that the rate of change in concentration of the        sodium acetate over the course of the gradient elution is about        25 mM/CV.        E240. The method of purifying a rAAV vector by AEX of any one of        E231-E239, wherein full capsids are eluted from the stationary        phase in a first elution peak; wherein full capsids are eluted        from the stationary phase in a first portion of a second elution        peak and/or wherein empty capsids are recovered in an AEX column        flow-through and/or in a last portion of a second elution peak.        E241. The method of purifying a rAAV vector by AEX of any one of        E231-E240, wherein the absorbance threshold is ≥0.5 mAU/mm path        length measured at 280 nm.        E242. The method of purifying a rAAV vector by AEX of any one of        E231-E241, wherein a volume of the at least one fraction of        eluate is equivalent to ≥⅓ of a CV.        E243. The method of purifying a rAAV vector by AEX of any one of        E231-E242, wherein the collecting at least one fraction of        eluate comprises collecting at least 10 fractions of eluate.        E244. The method of purifying a rAAV vector by AEX of any one of        E231-E242, wherein the pH of the at least one fraction of eluate        is adjusted to a pH of 6.8 to 7.6 (e.g., about pH 7.2).        E245. The method of purifying a rAAV vector by AEX of any one of        E231-E244, wherein the A260/A280 ratio of the at least one        fraction of eluate, the at least two fractions of eluate and/or        the pooled eluate is ≥1.25 (e.g., about 1.28 to 1.35).        E246. The method of purifying a rAAV vector by AEX of any one of        E231-E245, wherein the pooled eluate has a % VG column yield of        20% to 100% (e.g., 63+/−26%).        E247. The method of purifying a rAAV vector by AEX of any one of        E231-E246, wherein the pooled eluate has a % VG step yield of        31% to 66% (e.g., 47+/−11%).        E248. The method of purifying a rAAV vector by AEX of any one of        E231-E247, wherein at least one fraction of eluate and/or pooled        eluate is enriched for full capsids, and/or depleted of empty        capsids, as compared to the diluted and filtered affinity eluate        loaded onto the column.        E249. The method of purifying a rAAV vector by AEX of any one of        E231-E248, wherein a purified rAAV vector is produced.        E250. The method of purifying a rAAV vector by AEX of any one of        E231-E249, further comprising filtering the pooled eluate by a        method selected from the group consisting of viral filtration,        ultrafiltration/diafiltration (UF/DF), filtration through a 0.2        μm filter and a combination thereof, to produce a drug        substance.        E251. The method of purifying a rAAV vector by AEX of E250,        wherein the drug substance comprises 45% to 65% (e.g., 52+/−7%)        full capsids of the total capsids.        E252. The method of purifying a rAAV vector by AEX of E250 or        E251, wherein the drug substance comprises 19% to 37% (e.g.,        28+/−5%) intermediate capsids of total capsids.        E253. The method of purifying a rAAV vector by AEX of any one of        E250-E252, wherein the drug substance comprises 10% to 37%        (e.g., 20+/−7%) empty capsids of total capsids.        E254. The method of purifying a rAAV vector by AEX of any one of        E231-E253, wherein the rAAV vector comprises an AAV9 capsid        protein.        E255. A method of purifying a rAAV vector by AEX, the method        comprising a step of:    -   i) loading an affinity eluate comprising the rAAV vector to be        purified onto an AEX stationary phase (e.g., POROS™ 50 HQ) in a        column, wherein the eluate has been        -   a) diluted about 14.4 to 15.5 fold (e.g., about 15 fold)            with a buffer comprising about 200 mM histidine, about 200            mM Tris, about 0.5% P188, pH 8.7 to 9.0, and optionally        -   b) filtered through a 0.2 μm filter prior to loading onto            the stationary phase;    -   ii) performing gradient elution of a material from the        stationary phase in the column wherein about 20 CV of a first        gradient elution buffer (e.g., 100 mM Tris, 0.01% P188, pH 8.9),        a second gradient elution buffer (e.g., 500 mM sodium acetate,        100 mM Tris, 0.01% P188, pH 8.9) or a mixture of both are        applied to the stationary phase; wherein a concentration of the        sodium acetate is varied from 0 mM to 500 mM such that the rate        of change in concentration of the sodium acetate over the course        of the gradient elution is about 25 mM/CV;    -   iii) collecting about 10 fractions of eluate from the column        during the gradient elution when the A280 of the eluate is >0.5        mAU/mm path length, and wherein a volume of the at least one        fraction of eluate is equivalent to ≥⅓ of a CV;    -   iv) adjusting the pH of the about 10 fractions of eluate from        the column to a pH of 6.8 to 7.6 by addition of 14.3% to 15%        (eluate volume weight) of a solution comprising about 250 mM        sodium citrate, pH 3.5;    -   v) measuring an absorbance of the about 10 fractions of eluate        collected from the column and determining an A260/A280 ratio;        and/or    -   vi) combining at least two fractions of eluate collected from        the column to form a pooled eluate,    -   wherein the A260/A280 of each one of the at least two fractions        of eluate is ≥1.25;    -   wherein the pooled eluate is enriched for full capsids, and/or        depleted of empty capsids, as compared to the diluted, and        optionally filtered affinity eluate loaded onto the column; and    -   wherein a purified rAAV vector is produced.        E256. A method of purifying a rAAV vector by AEX of E255,        wherein the material eluted from the stationary phase comprises        the rAAV vector to be purified.        E257. A method of purifying a rAAV vector by AEX, the method        comprising a step of:    -   i) pre-use flushing comprising application of about 5 CV of        water for injection to an AEX stationary phase in a column;    -   ii) sanitizing comprising application of about 16 CV of a        solution comprising about 0.5 M NaOH to the AEX stationary phase        in the column, optionally by upward flow;    -   iii) regenerating comprising application of about 5 CV of a        solution comprising about 2 M NaCl, 100 mM Tris, pH 9 to the AEX        stationary phase in the column;    -   iv) equilibration comprising application of about 5 CV of a        solution comprising about 100 mM Tris, pH 9 to the AEX        stationary phase in the column;    -   v) equilibration comprising application of about 5 CV of an        equilibration buffer comprising about 100 mM Tris, 500 mM sodium        acetate, 0.01% P188, pH 8.9 to the AEX stationary phase in the        column;    -   vi) equilibration comprising application of about 5 CV of an        equilibration buffer comprising about 200 mM histidine, 200 mM        Tris, 0.5% P188, pH 8.8 to the AEX stationary phase in the        column;    -   vii) loading an affinity eluate comprising the rAAV vector to be        purified onto the AEX stationary phase in the column, optionally        wherein the eluate has been        -   a) diluted about 15 fold with a buffer comprising about 200            mM histidine, 200 mM Tris, 0.5% P188, pH 8.7 to 9.0, and            optionally        -   b) filtered through an 0.2 μm filter prior to loading onto            the stationary phase;    -   viii) equilibration comprising application of about 5 CV of an        equilibration buffer comprising about 100 mM Tris, 0.01% P188,        pH 8.9 to the AEX stationary phase in the column;    -   ix) performing gradient elution of a material from the        stationary phase in the column wherein about 20 CV of a first        gradient elution buffer (e.g., 100 mM Tris, P188, pH 8.9), a        second gradient elution buffer (e.g., 500 mM sodium acetate, 100        mM Tris, 0.01% P188, pH 8.9) or a mixture of both are applied to        the stationary phase; wherein a concentration of the sodium        acetate is varied from 0 mM to 500 mM such that the rate of        change in concentration of the sodium acetate over the course of        the gradient elution is about 25 mM/CV;    -   x) collecting about 10 fractions of eluate from the column        during a gradient elution when the A280 of the of the eluate is        ≥0.5 mAU/mm path length; and wherein a volume of the about 10        fractions of eluate is about ≥⅓ of a CV;    -   xi) adjusting a pH of the about 10 fractions of eluate to a pH        of 6.8 to 7.6 by addition of 14.3% to 15% (eluate volume weight)        of a solution comprising about 250 mM sodium citrate, pH 3.5;    -   xii) measuring of an absorbance of the about 10 factions of        eluate collected from the column and determining an A260/A280        ratio; and/or    -   xiii) combining at least two fractions of eluate collected from        the column to form a pooled eluate, wherein an A260/A280 of each        of the at least two fractions of eluate is ≥1.25;    -   wherein the pooled eluate is depleted of empty capsids, and/or        enriched for full capsids as compared to the affinity eluate        and/or the diluted, and optionally filtered affinity eluate;    -   wherein at least one of steps i) to ix) is performed at a linear        velocity of 270 to 330 cm/hr (e.g., about 300 cm/hr), a flow        rate of 1.5 to 2.0 L/min (e.g., about 1.8 L/min) or about 314        mL/min through a 1.3 L column and/or a residence time of about        3.5 to 4.5 min/CV (e.g., 4 min/CV); and/or wherein a purified        rAAV vector is produced.        E258. A method of purifying a rAAV vector by AEX of E257,        wherein the material eluted from the stationary phase comprises        the rAAV vector to be purified.        E259. A method of preparing a solution comprising a rAAV vector        for purification by AEX, the method comprising a step of:    -   i) diluting an affinity eluate about 15-fold with a dilution        solution comprising about 200 mM histidine, 200 mM Tris, 0.5%        P188, pH 8.8; and    -   ii) filtering the affinity eluate from step i) through a 0.2 μm        filter to produce a diluted and filtered affinity eluate;        wherein the pH of the diluted and filtered affinity eluate is        increased (e.g., to about 8.5 to 9.5) as compared to the pH of        the affinity eluate; and wherein the conductivity of the diluted        and filtered affinity eluate is decreased (e.g., to about 1.7        mS/cm to 3.3 mS/cm) as compared to the conductivity of the        affinity eluate.        E260. The method of preparing a solution comprising a rAAV        vector for purification by AEX of E259, wherein the diluted, and        optionally filtered affinity eluate is loaded on an AEX        stationary phase.        E261. The method of preparing a solution comprising a rAAV        vector for purification by AEX of E259 or E260, wherein the        affinity eluate is generated from affinity chromatography-based        purification of a rAAV vector produced in a vessel with a volume        of 250 L or 2000 L.        E262. The method of preparing a solution comprising a rAAV        vector for purification by AEX of any one of E259-E2561, wherein        a % VG dilution yield of the affinity eluate after dilution is        88%+/−36%.        E263. A method of preparing a stationary phase for use in a        method of purifying a rAAV vector by AEX, the method comprising        a step of:    -   i) pre-use flushing comprising application of ≥4.5 CV (e.g.,        about 5 CV) of water for injection to AEX stationary phase in a        column;    -   ii) sanitizing comprising application of about 14.4 to 17.6 CV        (e.g., about 16 CV) of a solution comprising about 0.1 M to 1.0        M (e.g., about 0.5 M) NaOH to the AEX stationary phase in the        column, optionally by upward flow; and/or    -   iii) regenerating comprising application of about 4.5 to 5.5 CV        (e.g., about CV) of a solution comprising about 1 M to 3 M NaCl,        50 mM to 150 mM Tris, pH 8.5 to 9.5 to the AEX stationary phase        in the column; optionally wherein at least one of steps i)-iii)        is performed at a linear velocity of 270 to 330 cm/hr (e.g.,        about 300 cm/hr), a flow rate of 1.5 to 2.0 L/min (e.g., about        1.8 L/min) and/or a residence time of 3.5 to 4.5 min/CV (e.g.,        about 4 min/CV).        E264. The method of preparing a stationary phase for use in a        method of purifying a rAAV vector by AEX of E263, further        comprising equilibration comprising application of about 5 CV of        one or more of the solutions comprising i) about 100 mM Tris, pH        9, ii) about 100 mM Tris, 500 mM sodium acetate, 0.01% P188, pH        8.9 and iii) about 200 mM histidine, 200 mM Tris, 0.5% P188, pH        8.8 to the AEX stationary phase in the column.        E265. The method of preparing a stationary phase for use in a        method of purifying a rAAV vector by AEX of E263 or E264,        wherein at least one step is performed prior to loading a        solution comprising the rAAV vector to be purified onto the        column.        E266. A purified rAAV vector prepared by a method of any one of        E1-E227 or E231-E258.        E267. A purified rAAV vector prepared by a method comprising a        step of:    -   i) loading a solution comprising the rAAV vector to be purified        onto an AEX stationary phase in a column;    -   ii) performing gradient elution of a material from the        stationary phase in the column wherein about 20 CV of a first        gradient elution buffer (e.g., 100 mM Tris, P188, pH 8.9), a        second gradient elution buffer (e.g., 500 mM sodium acetate, 100        mM Tris, 0.01% P188, pH 8.9) or a mixture of both are applied to        the stationary phase; wherein a concentration of a salt is        varied from 0 mM to 500 mM such that the rate of change in        concentration of the salt over the course of the gradient        elution is about 25 mM/CV;    -   iii) collecting at least one (e.g., about 10) fraction of eluate        from the column during a chromatography step (e.g., a gradient        elution) when the absorbance of a column flow-through reaches an        absorbance threshold (e.g., A280 is >0.5 mAU/mm path length);    -   iv) measuring an absorbance of the at least one fraction of        eluate collected from the column and determining an A260/A280        ratio; and/or    -   v) combining at least two fractions of eluate collected from the        column to form a pooled eluate comprising the purified rAAV        vector.        E268. The purified rAAV vector prepared by the method of E267,        wherein the material eluted from the stationary phase comprises        the rAAV vector to be purified.        E269. The purified rAAV vector prepared by the method of        E267-E268, wherein the salt is sodium acetate.        E270. The purified rAAV vector prepared by the method of any one        of E267-E269, wherein the solution is an affinity eluate that        has been a) diluted about 14.4 to fold (e.g., about 15 fold)        with a buffer comprising about 200 mM histidine, 200 mM Tris,        0.5% P188, pH 8.7 to 9.0, and optionally b) filtered through a        0.2 μm filter prior to loading onto the stationary phase.        E271. The purified rAAV vector prepared by the method of any one        of E267-E270, the method further comprising adjusting the pH of        the at least one (e.g., about 10) fraction of eluate from the        column to a pH of 6.8 to 7.6 by addition of 14.3% to 15% (eluate        volume weight) of a solution comprising about 250 mM sodium        citrate, pH 3.5.        E272. The purified rAAV vector of any one of E267-E271, wherein        the A260/A280 of each one of the at least two fractions of        eluate is ≥1.25; and wherein the pooled eluate is enriched for        full capsids, and/or depleted of empty capsids, as compared to        the diluted, and optionally filtered affinity eluate loaded onto        the column; and wherein the purified rAAV vector is produced.        E273. The purified rAAV vector of any one of E267-E272, wherein        the rAAV vector comprises an AAV9 capsid protein.        E274. A purified rAAV vector prepared by a method comprising a        step of:    -   i) loading a solution comprising the rAAV vector to be purified        onto an AEX stationary phase in a column;    -   ii) performing gradient elution of a material from the        stationary phase in the column wherein a percentage of a first        gradient elution buffer is varied in a manner inversely        proportional to variation in a percentage of a second gradient        elution buffer; iii) collecting at least one fraction of eluate        from the column during a chromatography step beginning when the        absorbance of a column-flow through reaches an absorbance        threshold;    -   iv) measuring an absorbance of the at least one fraction of        eluate collected from the column and determining an A260/A280        ratio; and/or    -   v) combining at least two fractions of eluate collected from the        column to form a pooled eluate comprising the purified rAAV        vector.        E275. The purified rAAV vector prepared by the method of E274,        wherein the material eluted from the stationary phase comprises        the rAAV vector to be purified        E276. The purified rAAV vector prepared by the method of E274 or        E275, wherein the rAAV vector comprises an AAV9 capsid protein.        E277. The purified rAAV vector prepared by the method of any one        of E274-E276, wherein the method further comprised filtering the        pooled eluate by a method selected from the group consisting of        viral filtration, ultrafiltration/diafiltration (UF/DF),        filtration through a 0.2 μm filter and a combination thereof, to        produce a drug substance.        E278. The purified rAAV vector prepared by the method of E277,        wherein the drug substance is used to make a drug product        suitable for administration to a human subject to treat a        disease, disorder or condition.        E279. The purified rAAV vector prepared by the method of E278,        wherein the disease, disorder or condition is DMD.        E280. A solution comprising a rAAV vector for purification by        AEX prepared by a method comprising a step of:    -   i) diluting a first solution (e.g., an affinity eluate) 2 to        25-fold (e.g., about with a buffer comprising 200 mM histidine,        200 mM Tris, 0.5% P188, pH 8.8;    -   and optionally    -   ii) filtering the first solution from step i) through a 0.2 μm        filter to produce a diluted and optionally filtered solution;        wherein the pH of the diluted and optionally filtered solution        is increased as compared to the pH of the first solution; and        wherein the conductivity of the diluted and optionally filtered        solution is decreased as compared to the conductivity of the        first solution.        E281. The solution comprising a rAAV vector for purification by        AEX of E280, wherein the first solution is an affinity eluate.        E282. The solution comprising a rAAV vector for purification by        AEX of E281, wherein the affinity eluate is generated from        affinity purification of a rAAV vector produced in a vessel        (e.g., a single use bioreactor) with a volume of 250 L or 2000        L.        E283. The solution comprising a rAAV vector for purification by        AEX of any one of E280-E282, wherein the pH of the diluted and        optionally filtered solution is 8.5 to 9.5.        E284. The solution comprising a rAAV vector for purification by        AEX of any one of E280-E283, wherein the conductivity of the        diluted and optionally filtered solution is 1.7 to 3.3 mS/cm.        E285. The solution comprising a rAAV vector for purification by        AEX of any one of E280-E284, wherein a % VG dilution yield of        the diluted and optionally filtered solution is 88%+/−36%.        E286. A solution comprising a rAAV vector for purification by        AEX prepared by a method comprising a step of:    -   i) diluting a first solution (e.g., an affinity eluate) 2 to        25-fold (e.g., 15-fold) with a dilution solution comprising        histidine, Tris, and P188; and optionally    -   ii) filtering the diluted first solution from step i) through a        0.2 μm filter to produce a diluted, and optionally filtered        solution; wherein the pH of the diluted, and optionally filtered        solution is increased as compared to the pH of the first        solution; and wherein the conductivity of the diluted, and        optionally filtered solution is decreased as compared to the        conductivity of the first solution.        E287. The solution comprising a rAAV vector for purification by        AEX prepared by a method of E280, wherein the dilution solution        comprises about 100 mM to 300 mM (e.g., about 200 mM) histidine,        100 mM to 300 mM (about 200 mM) Tris, 0.1% to 1.0% (about 0.5%)        P188, pH 8.5 to 9.5.        E288. The solution comprising a rAAV vector for purification by        AEX prepared by a method of E280 or E281, wherein the pH the        first solution is 3.0 to 4.4 prior to diluting, and optionally        filtering, and the pH of the first solution after diluting, and        optionally filtering, is 8.5 to 9.5 or 8.7 to 9.0 (e.g., 8.8,        9.0).        E289. The solution comprising a rAAV vector for purification by        AEX prepared by a method of any one of E280-E282, wherein        conductivity of the first solution is 5.0 to 7.0 mS/cm (e.g.,        5.5 to 6.5 mS/cm) prior to the step of diluting, and optionally        filtering, and the conductivity of the first solution after the        step of diluting, and optionally filtering, is 1.7 to 3.5 mS/cm,        1.8 to 2.8 mS/cm, or 2.2 to 2.6 mS/cm.        E290. The solution comprising a rAAV vector for purification by        AEX prepared by a method of any one of E280-E283, wherein the %        VG dilution yield of the diluted and optionally filtered first        solution is 35% to 100%.        E291. The solution comprising a rAAV vector for purification by        AEX prepared by a method of any one of E280-E28, wherein the        rAAV vector comprises an AAV9 or an AAV3B capsid protein, and        optionally, wherein the diluted and optionally filtered solution        is loaded on an AEX stationary phase.        E292. A method of regenerating an AEX stationary phase, the        method comprising a step of:    -   i) post-use sanitizing of the stationary phase comprising        application of 14.4 to 17.6 CV (e.g., about 16 CV) of a solution        comprising about 0.1 M to 1.0 M (e.g., about 0.5 M) NaOH to the        stationary phase, optionally by upward flow;    -   ii) regenerating the stationary phase comprising application of        4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising about        1 M to 3 M (e.g., about 2 M) NaCl, 50 mM to 150 mM (e.g., about        100 mM) Tris, pH 8.5 to 9.5 (e.g., about pH 9) to the stationary        phase;    -   iii) equilibration of the stationary phase comprising        application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution        comprising about 50 mM to 150 mM (e.g., about 100 mM) Tris, pH        8.5 to 9.5 (e.g., about pH 9) to the stationary phase;    -   iv) post-use flushing of the stationary phase comprising        application of ≥4.5 (e.g., about 5 CV) of water for injection to        the stationary phase; and/or    -   v) applying a storage solution to the stationary phase        comprising application 2.7 to 3.3 CV (e.g., about 3 CV) of a        solution comprising about 17.5% ethanol to the stationary phase,        optionally wherein at least one of steps i)-v) is performed at a        linear velocity of 270 to 330 cm/hr (e.g., about 300 cm/hr), a        flow rate of 1.5 to 2.0 L/min (e.g., about 1.8 L/min) through a        6.0 to 6.6 L (e.g., 6.4 L) column, or about 314 mL/min through a        1.3 L column, and/or a residence time of 3.5 to 4.5 min/CV        (e.g., about 4 min/CV).        E293. The method of regenerating an AEX stationary phase of        E292, wherein any one of steps i)-v) step follows a        chromatography elution step of a method of purifying a rAAV        vector by AEX.        E294. A regenerated AEX stationary phase prepared by a method        comprising a step of:    -   i) post-use sanitizing of the stationary phase comprising        application of 14.4 to 17.6 CV (e.g., about 16 CV) of a solution        comprising about 0.1 M to 1.0 M (e.g., about 0.5 M) NaOH to the        stationary phase, optionally by upward flow;    -   ii) regenerating the stationary phase comprising application of        4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising about        1 M to 3 M NaCl (e.g., about 2 M) NaCl, about 50 mM to 150 mM        (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about pH 9) to        the stationary phase;    -   iii) equilibration of the stationary phase comprising        application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution        comprising about 50 mM to 150 mM (e.g., about 100 mM) Tris, pH        8.5 to 9.5 (e.g., pH 9) to the stationary phase;    -   iv) post-use flushing of the stationary phase comprising        application of ≥4.5 (e.g., about 5 CV) of water for injection to        the stationary phase; and/or    -   v) applying a storage solution to the stationary phase        comprising application of 2.7 to 3.3 CV (e.g., about 3 CV) of a        solution comprising about 17% to 17.5% ethanol to the stationary        phase; optionally    -   wherein at least one of steps i)-v) step is performed at a        linear velocity of 270 to 330 cm/hr (e.g., about 300 cm/hr), a        flow rate of 1.5 to 2.0 L/min (e.g., about 1.8 L/min) through a        6.0 to 6.6 L (e.g., 6.4 L) column, or about 314 mL/min through a        1.3 L column, and/or a residence time of 3.5 to 4.5 min/CV        (e.g., about 4 min/CV).        E295. The regenerated AEX stationary phase of E288, wherein the        regenerated AEX stationary phase is used for purification of a        rAAV vector.        E296. A pharmaceutical composition comprising a purified rAAV        vector made by the method of any one of E1-E227 or E231-E258.        E297. A pharmaceutical composition comprising a purified rAAV        vector of E267-E279.        E298. Use of a rAAV vector purified according to the method of        any one of E1-E227 or E231-E258 in the manufacture of a        medicament for treating and/or preventing a disease, disorder or        condition.        E299. The use of E298, wherein the disease, disorder or        condition is DMD.        E300. The method of any one of E1-E182, wherein the rAAV vector        comprises a vector genome comprising a modified nucleic acid        encoding a deleted cooper-transporting ATPase2 (ATP7B) protein.        E301. The method of E300, wherein the modified nucleic acid        comprises a nucleic acid sequence encoding a deleted        cooper-transporting ATPase2 (ATP7B) protein comprising or        consisting of the amino acid sequence of SEQ ID NO:15.        E302. The method of E300 or E301, wherein the deleted        copper-transporting APTase2 comprises a deletion of        Heavy-Metal-Associated sited HMA 1, HMA 2, HMA 3 and HMA 4.        E303. The method of any one of E300-E302, wherein the vector        genome further comprises an α1-antitrypsin promoter, a        polyadenylation (polyA) signal sequence, a 5′ ITR and a 3′ ITR.        E304. The method of E303, wherein the α1-antitrypsin promoter        comprises or consists of the nucleic acid sequence of SEQ ID        NO:16.        E305. The method of E303, wherein the polyA signal sequence        comprises or consists of the nucleic acid sequence of SEQ ID        NO:17.        E306. The method of E303, wherein the 5′ ITR and the 3′ ITR are        AAV2 serotype ITRs.        E307. The method of any one of E1-E182, wherein the rAAV vector        comprises a VP1 polypeptide of AAV3B.        E308. The method of E307, wherein the VP1 polypeptide comprises        or consists of the amino acid sequence of SEQ ID NO:18.        E309. A method of purifying an rAAV vector by AEX, the method        comprising a step of:    -   i) loading a solution comprising the rAAV vector to be purified        onto an AEX stationary phase in a column;    -   ii) performing gradient elution of a material from the        stationary phase in the column wherein a percentage of a first        gradient elution buffer is varied in a manner inversely        proportional to variation in a percentage of a second gradient        elution buffer; wherein at the start of the gradient elution the        percentage of the first gradient elution buffer is about 75% to        about 100% and at the end of the gradient elution the percentage        of the second gradient elution buffer is about 60% to about        100%; and wherein the percentage of the second elution buffer        increases at a rate of about 2% to 5% per CV over the gradient        elution;    -   iii) collecting at least one fraction of eluate from the column        when performing the gradient elution beginning when the        percentage of the second gradient elution buffer is about 30% to        about 35%,    -   and wherein the at least one fraction of eluate comprises the        rAAV vector to be purified.        E310. The method of purifying an rAAV vector by AEX of E309,        wherein the solution comprising the rAAV vector is an affinity        eluate that has been diluted about fold with a buffer comprising        histidine, Tris and P188.        E311. The method of purifying an rAAV vector by AEX of E309 or        E310, wherein the first gradient elution buffer comprises 50 mM        to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g.,        about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9) and/or the second        gradient elution buffer comprises 400 mM to 600 mM (e.g., about        500 mM) sodium acetate, 50 mM to 150 mM (e.g., about 100 mM)        Tris, 0.005% to (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g.,        pH 8.9).        E312. The method of purifying an rAAV vector by AEX of any one        of E309-E311, wherein collecting at least one fraction of eluate        from the column comprises collecting the at least one fraction        of eluate into a vessel comprising about 0.01 CV to 0.1 CV        (e.g., about 0.066 CV) of a solution comprising 200 mM to 300 mM        (e.g., about 250 mM) sodium citrate, pH 3.0 to 4.0 (e.g., about        3.5).        E313. The method of purifying a rAAV vector by AEX of any one of        E310-E312, wherein the at least one fraction of eluate is        enriched for full capsids, and/or depleted of empty capsids, as        compared to the affinity eluate; optionally wherein the rAAV        vector is a rAAV3B vector; and optionally wherein the AEX        stationary phase is POROS™ 50 HQ.        E314. The method of purifying a rAAV vector by AEX of any one of        E309-E313, wherein the collecting at least one fraction of        eluate from the column when performing the gradient elution ends        when the percentage of the second gradient elution buffer is        about 50% to about 55%.        E315. A method of purifying an rAAV vector by AEX, the method        comprising a step of: i) loading a solution comprising the rAAV        vector to be purified onto an AEX stationary phase in a column;    -   ii) performing gradient elution of a material from the        stationary phase in the column wherein a percentage of a first        gradient elution buffer is varied in a manner inversely        proportional to variation in a percentage of a second gradient        elution buffer; and    -   iii) collecting at least one fraction of eluate from the column        during the gradient elution beginning when the absorbance of a        column flow-through reaches an absorbance threshold, and wherein        the at least one fraction of eluate comprises the rAAV vector to        be purified.        E316. The method of purifying a rAAV vector by AEX of E315,        wherein the method further comprises measuring an absorbance of        the at least one fraction of eluate collected from the column        and determining an A260/A280 ratio.        E317. The method of purifying a rAAV vector by AEX of E315 or        E316, wherein the solution comprising the rAAV vector to be        purified is diluted about 2-fold to 25-fold (e.g., 15-fold) with        a dilution solution comprising histidine, Tris and P188, and        optionally filtered prior to application to the stationary        phase.        E318. The method of purifying a rAAV vector by AEX of any one of        E315-E317, wherein the solution is an affinity eluate.        E319. The method of purifying a rAAV vector by AEX of E315-E318,        wherein a pH of the diluted, and optionally filtered affinity        eluate is increased as compared to a pH of the solution; and        wherein a conductivity of the diluted, and optionally filtered        affinity eluate is decreased as compared to a conductivity of        the solution.        E320. The method of purifying a rAAV vector by AEX of any one of        E315-E319, wherein the first gradient elution buffer comprises        about 50 mM to about 150 mM Tris, about 0.005% to about 0.015%        P188 and has a pH of about pH 8.5 to 9.5; wherein the second        gradient elution buffer comprises about 400 mM to about 600 mM        sodium acetate, about 50 mM to about 150 mM Tris, about 0.005%        to about P188 and has a pH of about pH 8.5 to 9.5; and wherein        10 to 60 column volumes (CV) (e.g., about 20 CV, about 37.5 CV)        of the first gradient elution buffer, the second gradient        elution buffer or a mixture of both are applied to the        stationary phase during the gradient elution.        E321. The method of purifying a rAAV vector by AEX of any one of        E315-E320, wherein at a start of the gradient elution the        percentage of the first gradient elution buffer is 50%-100% and        at an end of the gradient elution the percentage of the second        gradient elution buffer is 50%-100% and wherein the percentage        of the second elution buffer increases at a rate of about 2% to        5% per CV over the gradient elution.        E322. The method of purifying a rAAV vector by AEX of any one of        E315-E321, wherein a concentration of sodium acetate of the        first gradient elution buffer, the second gradient elution        buffer or the mixture of both increases continuously during the        gradient elution; and wherein the concentration of the sodium        acetate increases at a rate of about 10 mM/CV to 50 mM/CV (e.g.,        about 10 mM/CV, about 25 mM/CV) over the gradient elution.        E323. The method of purifying a rAAV vector by AEX of any one of        E315-E322, wherein full capsids are eluted from the stationary        phase in a first elution peak and/or in a first portion of a        second elution peak during the gradient elution.        E324. The method of purifying a rAAV vector by AEX of any one of        E315-E323, wherein empty capsids are recovered in the column        flow-through, in a first elution peak and/or in a last portion        of a second elution peak during the gradient elution.        E325. The method of purifying a rAAV vector by AEX of any one of        E315-E324, wherein an absorbance of the at least one fraction of        eluate is measured at 280 nm, and wherein optionally, an        absorbance threshold is ≥0.5 mAU/mm path length measured at 280        nm.        E326. The method of purifying a rAAV vector by AEX of any one of        E315-E325, wherein a volume of the at least one fraction of        eluate is equivalent to ⅛ of a CV to 10 CV, e.g., ⅛ of a CV, ¼        of a CV, ⅓ of a CV, ½ of a CV, ¾ of a CV, 1 CV, 2 CV, 3 CV, 4        CV, 5 CV, 6 CV, 7 CV, 8 CV, 9 CV, 10 CV or more of a CV, and        optionally, wherein an A260/A280 ratio of the at least one        fraction of eluate is ≥ to 1.25.        E327. The method of purifying a rAAV vector by AEX of any one of        E315-E326, wherein at least 2, at least 3, at least 4, at least        5, at least 6, at least 7, at least 8, at least 9, at least 10,        at least 11, at least 12, at least 13, at least 14, at least 15,        at least 16, at least 17, at least 18, at least 19, at least 20,        at least 21, at least 22, at least 23, at least 24, at least 25,        or more, fractions of eluate are collected.        E328. The method of purifying a rAAV vector by AEX of any one of        E315-E327, wherein the method further comprises combining at        least two fractions of eluate collected from the column, each        having an A260/A280 ratio of ≥0.98 or ≥1.0 to form a pooled        eluate comprising the rAAV vector.        E329. The method of purifying a rAAV vector by AEX of E328,        wherein the pooled eluate has a % VG column yield of 20% to 100%        (e.g., 63+/−26%), a % VG step yield of 31% to 66% (e.g.,        47+/−11%) and/or an A260/A280 ratio of ≥1.0.        E330. The method of purifying a rAAV vector by AEX of E328 or        E329, wherein the pooled eluate is enriched for full capsids,        and/or depleted of empty capsids, as compared to the solution        loaded onto the column.        E331. The method of purifying a rAAV vector by AEX of any one of        E315-E330, wherein a purified rAAV vector is produced.        E332. The method of purifying a rAAV vector by AEX of any one of        E328-E331, further comprising filtering the pooled eluate by a        method selected from the group consisting of viral filtration,        ultrafiltration/diafiltration (UF/DF), filtration through a 0.2        μm filter and a combination thereof, to produce a drug        substance.        E333. The method of purifying a rAAV vector by AEX of E332,        wherein the drug substance comprises: i) 45% to 65% (e.g.,        52+/−7%) full capsids of total capsids; ii) 19% to 37% (e.g.,        28+/−5%) intermediate capsids of total capsids; and/or iii) 10%        to 37% (e.g., 20+/−7%) empty capsids of total capsids.        E334. The method of purifying a rAAV vector by AEX of E332-E333,        wherein the drug substance is enriched for full capsids, and/or        depleted of empty capsids, as compared to the solution loaded        onto the column.        E335. The method of purifying a rAAV vector by AEX of any one of        E315-E334, wherein the rAAV vector comprises an AAV capsid        protein from an AAV serotype selected from the group consisting        of AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8,        AAV9, AAV10, AAVrh10, AAVrh74, AAV12, AAV2i8, NP4, NP22, NP66,        AAVDJ, AAVDJ/8, AAVDJ/9, AAVLK03, AAV1.1, AAV2.5, AAV6.1,        AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO:5 of WO 2015/013313),        RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAV hu.26,        AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV2i8, AAV29G,        AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N,        AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7,        AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13,        AAVHSC14 and AAVHSC15.        E336. The method of purifying a rAAV vector by AEX of any one of        E315-E335, wherein the rAAV vector comprises an AAV9 capsid        protein and a transgene comprising the nucleic acid of SEQ ID        NO:1.        E337. The method of purifying a rAAV vector by AEX of any one of        E315-E336, wherein the rAAV vector comprises an AAV3B capsid        protein and a transgene comprising a nucleic acid encoding the        amino acid sequence of SEQ ID NO:15.        E338. A method of preparing a solution comprising a rAAV vector        for purification by AEX, the method comprising a step of:    -   i) diluting a first solution 2 to 25-fold (e.g., 15-fold) with a        dilution solution comprising histidine, Tris and P188; and        optionally    -   ii) filtering the first solution from step i) through a filter        to produce a diluted, and optionally filtered solution;        wherein the pH of the diluted, and optionally filtered solution        is increased as compared to the pH of the first solution; and        wherein the conductivity of the diluted, and optionally filtered        solution is decreased as compared to the conductivity of the        first solution.        E339. The method of preparing a solution comprising a rAAV        vector for purification by AEX of E338, wherein the first        solution comprising the rAAV vector is selected from the group        consisting of an affinity eluate, a supernatant from a cell        lysate and a post-harvest solution.        E340. The method of preparing a solution comprising a rAAV        vector for purification by AEX of E338 or E339, wherein the        dilution solution comprises about 100 mM to 300 mM (e.g., about        200 mM) histidine, 100 mM to 300 mM (e.g., about 200 mM) Tris,        0.1% to 1.0% (e.g., about 0.5%) P188 and has a pH of pH 8.5 to        9.5.        E341. The method of preparing a solution comprising a rAAV        vector for purification by AEX of any one of E338-E340,        wherein i) a pH of the diluted, and optionally filtered solution        is 8.5 to 9.5; ii) a conductivity of the diluted, and optionally        filtered solution is 1.7 to 3.3 mS/cm; and/or iii) a % VG        dilution yield of the diluted solution is 35% to 100%.        E342. The method of preparing a solution comprising a rAAV        vector for purification by AEX of any one of E338-E341, wherein        the rAAV vector comprises an AAV9 capsid protein or an AAV3B        capsid protein.        E343. A purified rAAV vector prepared by a method comprising a        step of:    -   i) loading a solution comprising the rAAV vector to be purified        onto an AEX stationary phase in a column;    -   ii) performing gradient elution of a material from the        stationary phase in the column wherein a first gradient elution        buffer, a second gradient elution buffer or a mixture of both        are applied to the stationary phase and a concentration of a        salt is varied from 0 mM to 500 mM such that the rate of        increase in concentration of the salt over the course of the        gradient elution is about 10 mM/CV to 50 mM/CV (e.g., about 25        mM/CV);    -   iii) collecting at least one fraction of eluate from the column        during gradient elution beginning when absorbance of a column        flow-through reaches an absorbance threshold; and/or    -   vi) measuring an absorbance of the at least one fraction of        eluate collected from the column and determining an A260/A280        ratio.        E344. The purified rAAV vector prepared by the method of E343,        wherein the method further comprises combining at least two        fractions of eluate collected from the column when the A260/A280        ratio is ≥1.0 to form a pooled eluate comprising the purified        rAAV vector.        E345. The purified rAAV vector prepared by the method of E343 or        E344, wherein the salt is sodium acetate.        E346. The purified rAAV vector prepared by the method of any one        of E343-E345, wherein the rAAV vector comprises an AAV9 capsid        protein or an AAV3B capsid protein.        E347. The purified rAAV vector prepared by the method of any one        of E343-E346, wherein the solution comprising the rAAV vector is        an affinity eluate that has been diluted and optionally filtered        prior to loading onto the stationary phase.        E348. The purified rAAV vector prepared by the method of any one        of E343-E347, wherein the material eluted from the stationary        phase comprises the rAAV vector.        E349. The purified rAAV vector prepared by the method of any one        of E344-E348, wherein the method further comprises filtering the        pooled eluate by a method selected from the group consisting of        viral filtration, ultrafiltration/diafiltration (UF/DF),        filtration through a 0.2 μm filter and a combination thereof, to        produce a drug substance.        E350. The purified rAAV vector prepared by the method of E349,        wherein the drug substance is used to make a drug product        suitable for administration to a human subject to treat a        disease, disorder or condition.        E351. The purified rAAV vector prepared by the method of E350,        wherein the disease, disorder or condition is DMD or Wilson's        disease, and optionally wherein the rAAV vector comprises a        nucleic acid encoding the amino acid sequence of SEQ ID NO:2 or        SEQ ID NO:15.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts the elution phases of exemplary AEX chromatogramsgenerated using four elution salts on a 1 mL POROS™ 50 HQ column. A₂₆₀trace is shown by a dashed line, while A₂₈₀ and conductivity traces areshown by solid lines. Solid bars indicate elution fractions that wereused to form pools.

FIG. 2 depicts exemplary SEC A₂₆₀/A₂₈₀ of AEX elution fractionsgenerated using four elution salts on a 1 mL POROS™ 50 HQ column.

FIG. 3 depicts an exemplary AEX chromatogram generated using a sodiumacetate 9-step wash and elution carried out on a 5.1 mL POROS™ 50 HQcolumn. A₂₆₀ trace is shown by a dashed line, while A₂₈₀ andconductivity traces are shown by solid lines. Wash (W), elution (E),strip, and regeneration (Regen.) fractions are indicated consistent withTable 5 and Table 6.

FIG. 4A depicts an exemplary AEX chromatogram generated using a sodiumacetate step elution run with a 600 cm/hr elution, 5.1×10¹³ vectorgenome/mL resin challenge (VG/mL resin, as measured by qPCR of the ITRsequences), and 57 mM Sodium acetate wash carried out on a 5.1 mL POROS™50 HQ column. A₂₆₀ trace is shown by a dashed line, while A₂₈₀ andconductivity traces are shown by solid lines. FIG. 4B depicts amagnified view of the chromatogram at the wash, elution and strip phasesof the AEX run.

FIG. 5 depicts an exemplary method of in-line mixing of an AAV9 affinityeluate with 100 mM Tris, pH 9 to generate an AEX load (also referred toherein as a diluted affinity eluate). Fluids were delivered to theY-connector with peristaltic pumps.

FIG. 6 depicts exemplary pH, conductivity, Z-Average, and aggregation(given as + or −) of an AAV9 affinity eluate diluted with 100 mM Tris,pH 9.

FIG. 7A and FIG. 7B depict exemplary % vector genome (VG) yield foraffinity eluates diluted 5, 9, or 25-fold with 200 mM histidine, 200 mMTris, X % (w/v) P188, pH 8.8, where X is 0.01%, 0.05%, 0.2%, and 0.5%,followed by filtration. A contour plot of % VG yield (post dilution andfiltration) as a function of conductivity (controlled by dilutionfactor) and P188 concentration is depicted in FIG. 7A. One way ANOVAanalyses of % VG yield (post dilution and filtration) as a function ofP188 concentration or conductivity are depicted in FIG. 7B. Data is alsopresented in Table 13.

FIG. 8A depicts an exemplary chromatogram generated using the optimizedAEX process. FIG. 8B depicts a zoom-in of AEX sodium acetate gradientelution, with fractions numbered 1-14, consistent with Table 15. A₂₆₀trace is given in dashed line, while A₂₈₀ and conductivity traces aregiven in solid lines.

FIG. 9 depicts exemplary SEC A₂₆₀/A₂₈₀ values of chromatographicfractions generated using the optimized AEX method on 0%, 20%, 40%, 60%,80%, and 100% Null affinity pools. Flow-through is abbreviated as F/T.

FIG. 10 depicts the elution phase of an exemplary 250 L SUB AEXchromatogram from Batch 250L-4, run on a 10 cm inner diameter (ID)×16 cmbed height (BH), 1.3 L POROS™ 50 HQ column. A₂₆₀ trace is shown by adashed line, while A₂₈₀ and conductivity traces are shown by solidlines.

FIG. 11 depicts the elution phase of an exemplary 2000 L SUB Scale AEXchromatogram, batch 2000 L-4, run on a 20 cm ID×20.5 cm BH, 6.4 L POROS™50 HQ column. A₂₆₀ trace is shown by a dashed line, while A₂₈₀ andconductivity traces are shown by solid lines.

FIG. 12 depicts and exemplary chromatogram using the optimized AEXprocess for the purification of an AAV3B vector. A₂₆₀ trace is shown bya solid line, while A₂₈₀ trace is shown by a dashed line.

DESCRIPTION 1. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which this invention belongs. The terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting of the invention. As used in the description of theinvention and the appended claims, the singular forms “a,” “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. The following terms have themeanings given:

As used herein, the term “about,” or “approximately” refers to ameasurable value such as an amount of the biological activity, length ofa polynucleotide or polypeptide sequence, content of G and Cnucleotides, codon adaptation index, number of CpG dinucleotides, dose,time, temperature, and the like, and is meant to encompass variations of25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 0.5% or even 0.1%, in either direction (greater thanor less than) of the specified amount unless otherwise stated, otherwiseevident from the context, or except where such number would exceed 100%of a possible value.

As used herein, the term “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

As used herein, the terms “adeno-associated virus” and/or “AAV” refer toa parvovirus with a linear single-stranded DNA genome and variantsthereof. The term covers all subtypes and both naturally occurring andrecombinant forms, except where required otherwise.

The canonical AAV wild-type genome comprises 4681 bases (Berns andBohenzky (1987) Advances in Virus Research 32:243-307) and includesterminal repeat sequences (e.g., inverted terminal repeats (ITRs)) ateach end which function in cis as origins of DNA replication and aspackaging signals for the virus. The genome includes two large openreading frames, known as AAV replication (“AAV rep” or “rep”) and capsid(“AAV cap” or “cap”) genes, respectively. AAV rep and cap may also bereferred to herein as AAV “packaging genes.” These genes code for theviral proteins involved in replication and packaging of the viralgenome.

In wild type AAV, three capsid genes VP1, VP2 and VP3 overlap each otherwithin a single open reading frame and alternative splicing leads toproduction of VP1, VP2 and VP3 capsid proteins (Grieger and Samulski(2005) J. Virol. 79(15):9933-9944). A single P40 promoter allows allthree capsid proteins to be expressed at a ratio of about 1:1:10 forVP1, VP2, VP3, respectively, which complements AAV capsid production.More specifically, VP1 is the full-length protein, with VP2 and VP3being increasingly shortened due to increasing truncation of theN-terminus. A well-known example is the capsid of AAV9 as described inU.S. Pat. No. 7,906,111, wherein VP1 comprises amino acid residues 1 to736 of SEQ ID NO:123, VP2 comprises amino acid residues 138 to 736 ofSEQ ID NO:123, and VP3 comprises amino acid residues 203 to 736 of SEQID NO:123. As used herein, the term “AAV Cap” or “cap” refers to AAVcapsid proteins VP1, VP2 and/or VP3, and variants and analogs thereof. Asecond open reading frame of the capsid gene encodes an assembly factor,called assembly-activating protein (AAP), which is essential for thecapsid assembly process (Sonntag et al. (2011) J. Virol.

At least four viral proteins are synthesized from the AAV rep gene—Rep78, Rep 68, Rep 52 and Rep 40—named according to their apparentmolecular weights. As used herein, “AAV rep” or “rep” means AAVreplication proteins Rep 78, Rep 68, Rep 52 and/or Rep 40, as well asvariants and analogs thereof. As used herein, rep and cap refer to bothwild type and recombinant (e.g., modified chimeric, and the like) repand cap genes as well as the polypeptides they encode. In someembodiments, a nucleic acid encoding a rep will comprise nucleotidesfrom more than one AAV serotype. For instance, a nucleic acid encoding arep protein may comprise nucleotides from an AAV2 serotype andnucleotides from an AAV3 serotype (Rabinowitz et al. (2002) J. Virology76(2):791-801).

As used herein the terms “recombinant adeno-associated virus vector,”“rAAV” and/or “rAAV vector” refer to an AAV capsid comprising a vectorgenome. The vector genome comprises a polynucleotide sequence that isnot, at least in part, derived from a naturally-occurring AAV (e.g., aheterologous polynucleotide not present in wild type AAV), and the repand/or cap genes of the wild type AAV genome have been removed from thevector genome. Where the rep and/or cap genes of the AAV have beenremoved (and/or ITRs from an AAV have been added or remain), the nucleicacid within the AAV is referred to as the “vector genome.”

Therefore, the term rAAV vector encompasses both a rAAV viral particlethat comprises a capsid but does not comprise a complete AAV genome;instead the recombinant viral particle can comprise a heterologous,i.e., not originally present in the capsid, nucleic acid, hereinafterreferred to as a vector genome. Thus, a “rAAV vector genome” (or “vectorgenome”) refers to a heterologous polynucleotide sequence (including atleast one ITR) that may, but need not, be contained within an AAVcapsid. A rAAV vector genome may be double-stranded (dsAAV),single-stranded (ssAAV) or self-complementary (scAAV). Typically, avector genome comprises a heterologous (to the original AAV from whichit is derived) nucleic acid often encoding a therapeutic transgene, agene editing nucleic acid, and the like.

As used herein, the terms “rAAV vector,” “rAAV viral particle” and/or“rAAV vector particle” refer to an AAV capsid comprised of at least oneAAV capsid protein (though typically all of the capsid proteins, e.g.,VP1, VP2 and VP3, or variant thereof, of a AAV are present) andcontaining a vector genome comprising a heterologous nucleic acidsequence. These terms are to be distinguished from an “AAV viralparticle” or “AAV virus” that is not recombinant wherein the capsidcontains a virus genome encoding rep and cap genes and which AAV virusis capable of replicating if present in a cell also comprising a helpervirus, such as an adenovirus and/or herpes simplex virus, and/orrequired helper genes therefrom. Thus, production of a rAAV vectorparticle necessarily includes production of a recombinant vector genomeusing recombinant DNA technologies, as such, which vector genome iscontained within a capsid to form a rAAV vector, rAAV viral particle, ora rAAV vector particle.

The genomic sequences of various serotypes of AAV, as well as thesequences of the inverted terminal repeats (ITRs), rep proteins, andcapsid subunits are known in the art. Such sequences may be found in theliterature or in public databases such as GenBank. See, e.g., GenBankAccession Numbers NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2),AF043303 (AAV2), NC_001729 (AAV3), AF028705.1 (AAV3B), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF028704 (AAV6), AF513851 (AAV7),AF513852 (AAV8), NC_006261 (AAV8), AY530579 (AAV9), AY631965 (AAV10),AY631966 (AAV11), and DQ813647 (AAV12); the disclosures of which areincorporated by reference herein. See also, e.g., Srivistava et al.(1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823;Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999)J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu etal. (1996) Virology 221:208; Shade et al. (1986) J. Virol. 58:921; Gaoet al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004)Virology 33:375-383; international patent publications WO 00/28061, WO99/61601, WO 98/11244; WO 2013/063379, WO 2014/194132, WO 2015/121501;and U.S. Pat. Nos. 6,156,303 and 7,906,111.

As used herein, the term “associated with” refers to with one another,if the presence, level and/or form of one is correlated with that of theother. For example, a particular entity (e.g., polypeptide, geneticsignature, metabolite, microbe, etc.) is considered to be associatedwith a particular disease, disorder, or condition, if its presence,level and/or form correlates with incidence of and/or susceptibility tothe disease, disorder, or condition (e.g., across a relevantpopulation). In some embodiments, two or more entities are physically“associated” with one another if they interact, directly or indirectly,so that they are and/or remain in physical proximity with one another.In some embodiments, two or more entities that are physically associatedwith one another are covalently linked to one another; in someembodiments, two or more entities that are physically associated withone another are not covalently linked to one another but arenon-covalently associated, for example, by means of hydrogen bonds, vander Waals interaction, hydrophobic interactions, magnetism, and acombination thereof.

As used herein, the term “coding sequence” or “nucleic acid encoding”refers to a nucleic acid sequence which encodes a protein or polypeptideand denotes a sequence which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vitro or in vivowhen placed under the control of (operably linked to) appropriateregulatory sequences. The boundaries of a coding sequence are generallydetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A coding sequence can include,but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomicDNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNAsequences.

As used herein, the term “chimeric” refers to a viral capsid orparticle, with capsid or particle sequences from different parvoviruses,preferably different AAV serotypes, as described in Rabinowitz et al.,U.S. Pat. No. 6,491,907, the disclosure of which is incorporated in itsentirety herein by reference. See also Rabinowitz et al. (2004) J.Virol. 78(9):4421-4432. In some embodiments, a chimeric viral capsid isan AAV2.5 capsid which has the sequence of the AAV2 capsid with thefollowing mutations: 263 Q to A; 265 insertion T; 705 N to A; 708 V toA; and 716 T to N. The nucleotide sequence encoding such capsid isdefined as SEQ ID NO: 15 as described in WO 2006/066066. Other preferredchimeric AAV capsids include, but are not limited to, AAV2i8 describedin WO 2010/093784, AAV2G9 and AAV8G9 described in WO 2014/144229, andAAV9.45 (Pulicherla et al. (2011) Molecular Therapy 19(6):1070-1078),AAV-NP4, NP22 and NP66, AAV-LK0 through AAV-LK019 described in WO2013/029030, RHM4-1 and RHM15_1 through RHM5_6 described in WO2015/013313, AAVDJ, AAVDJ/8, AAVDJ/9 described in WO 2007/120542.

As used herein, the term “eluate” refers to fluid exiting from achromatography stationary phase (e.g., a monolith, membrane, resin,media) (e.g., “eluting from the stationary phase”) comprised of mobilephase and material that passed through the stationary phase or wasdisplaced from the stationary phase. In some embodiments, a stationaryphase includes, for example, a monolith, a membrane, a resin or a media.The mobile phase may be a solution that has been loaded onto a columnand has flowed through the column (i.e., “flow-through fraction”); anequilibration solution (e.g. an equilibration buffer); an isocraticelution solution; a gradient elution solution; a solution forregenerating a stationary phase; a solution for sanitizing a stationaryphase; a solution for washing; and combinations thereof.

As used herein, the term “flanked,” refers to a sequence that is flankedby other elements and indicates the presence of one or more flankingelements upstream and/or downstream, i.e., 5′ and/or 3′, relative to thesequence. The term “flanked” is not intended to indicate that thesequences are necessarily contiguous. For example, there may beintervening sequences between a nucleic acid encoding a transgene and aflanking element. A sequence (e.g., a transgene) that is “flanked” bytwo other elements (e.g., ITRs), indicates that one element is located5′ to the sequence and the other is located 3′ to the sequence; however,there may be intervening sequences there between.

As used herein, the term “flocculation” refers to the process by whichfine particulates are caused to clump together into a floc. The fineparticles may include proteins, nucleic acids, cellular fragmentsresulting from lysis of host cells. In some embodiments, a floc thatforms in a liquid phase may float to the top of the liquid (creaming),settle to the bottom (sedimentation) of the liquid or be filtered fromthe liquid phase.

As used herein, the term “fragment” refers to a material or entity thathas a structure that includes a discrete portion of the whole but lacksone or more moieties found in the whole. In some embodiments, a fragmentconsists of a discrete portion. In some embodiments, a fragment consistsof or comprises a characteristic structural element or moiety found inthe whole. In some embodiments, a polymer fragment comprises, orconsists of, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or moremonomeric units (e.g., amino acid residues, nucleotides) found in thewhole polymer.

rAAV vectors are referred to as “full,” a “full capsid,” “full vector”or a “fully packaged vector” when the capsid contains a complete, oressentially complete, vector genome, including a transgene. Duringproduction of rAAV vectors by host cells, vectors may be produced thathave less packaged nucleic acid than the full capsids and contain, forexample a partial or truncated vector genome. These vectors are referredto as “intermediates,” an “intermediate capsid,” a “partial” or a“partially packaged vector.” An intermediate capsid may also be a capsidwith an intermediate sedimentation rate, that is a sedimentation ratebetween that of full capsids and empty capsids, when analyzed byanalytical ultracentrifugation. Host cells may also produce viralcapsids that do not contain any detectable nucleic acid material. Thesecapsids are referred to as “empty(s),” or “empty capsids.” Full capsidsmay be distinguished from empty capsids based on A260/A280 ratiosdetermined by SEC-HPLC, whereby the A260/A280 ratios have beenpreviously calibrated against capsids (i.e., full, intermediate andempty) isolated by analytical ultracentrifugation. Other methods knownin the art for the characterization of capsids include CryoTEM,capillary isoelectric focusing and charge detection mass spectrometry.Calculated isoelectric points of ˜6.2 and ˜5.8 for empty and full AAV9capsids, respectively have been reported (Venkatakrishnan et al., J.Virology (2013) 87.9:4974-4984).”

As used herein, the term “null capsid” refers to a capsid producedintentionally to lack a vector genome. Such null a capsid may beproduced by transfection of a host cell with a rep/cap and a helperplasmid, but not a plasmid that comprises the transgene cassettesequence, also known as a vector plasmid.

As used herein, the term “functional” refers to a biological molecule ina form in which it exhibits a property and/or activity by which it ischaracterized. A biological molecule may have two functions (i.e.,bifunctional) or many functions (i.e., multifunctional).

As used herein, the term “gene” refers to a polynucleotide containing atleast one open reading frame that is capable of encoding a particularpolypeptide or protein after being transcribed and translated. “Genetransfer” or “gene delivery” refers to methods or systems for reliablyinserting foreign DNA into host cells. Such methods can result intransient expression of non-integrated transferred DNA, extrachromosomalreplication and expression of transferred replicons (e.g. episomes),and/or integration of transferred genetic material into the genomic DNAof host cells.

As used herein, the term “gradient elution” refers to application of amixture of at least two different solutions with different pH,conductivity and/or modifier concentration to a chromatographystationary phase (including e.g., monolith, media, resin, membrane) thatare gradually changed over the course of the elution. A gradient elutionmay be linear or non-linear. In contrast, during an isocratic elution,the chromatography mobile phase composition is constant, and during a“step elution,” the chromatography mobile phase composition changes in astepwise manner. Over the course of the gradient elution, a percentageof a first solution is continuously varied in a manner inverselyproportional to a percentage of a second solution. For example, at thestart of a gradient elution, the percentage of gradient elution buffer A(e.g., a first gradient elution buffer) in the mixture is 100% and thepercentage of gradient elution buffer B (e.g., a second gradient elutionbuffer) in the mixture is 0% such that a continuously varying gradientin the pH, conductivity and/or modifier concentration (increasing ordecreasing, depending on the embodiment) is created as the solutions aremixed and flow through the stationary phase. In some embodiments, aconcentration of a salt, such as sodium acetate, will change at aconstant rate over the volume of a linear gradient. For example, for a 1mL column with a 20 mL linear gradient (i.e., 20 CV), operating at aconstant flow rate of 1 mL/minute, the salt concentration will change ata rate of 5% per minute. In some embodiments, rAAV capsids (e.g., full,intermediate, empty) are bound to a stationary phase during loading of asolution comprising the rAAV capsid to be purified onto an AEXstationary phase. During a gradient elution, as a percentage of buffer Bincreases, such that the concentration of a salt increases (e.g., Sodiumacetate) full rAAV vectors are preferentially released (eluted) from thestationary phase, and empty capsids are preferentially retained on thestationary phase. Empty capsids are released in greater amounts as thepercentage of buffer B further increases. Elution of full rAAV vectorfrom the stationary phase can be monitored during gradient elution bymeasuring A260 and A280 of the eluate, such that an increase in theratio of A260/A280 is indicative of an increase in the percentage offull rAAV vector in the eluate, and conversely, a decrease in theA260/A280 ratio is indicative of a decrease in the percentage of fullrAAV vector and an increase in the percentage of empty capsids. In someembodiments, an absorbance of at least one fraction of eluate ismeasured using a method such as analytical size exclusion chromatography(SEC) in a high performance liquid chromatography (HPLC) system, on-lineUV trace, off-line UV methods, etc., and wherein the absorbance ismeasured at one or more wavelengths (e.g., 260 nm and/or 280 nm).

As used herein, the term “heterologous” refers to a nucleic acidinserted into a vector (e.g., rAAV vector) for purposes of vectormediated transfer/delivery of the nucleic acid into a cell. Heterologousnucleic acids are typically distinct from the vector (e.g., AAV) nucleicacid, that is, the heterologous nucleic acid is non-native with respectto the viral (e.g., AAV) nucleic acid. Once transferred or deliveredinto a cell, a heterologous nucleic acid, contained within a vector, canbe expressed (e.g., transcribed and translated if appropriate).Alternatively, a transferred or delivered heterologous nucleic acid in acell, contained within the vector, need not be expressed. Although theterm “heterologous” is not always used herein in reference to a nucleicacid, reference to a nucleic acid even in the absence of the modifier“heterologous” is intended to include a heterologous nucleic acid. Forexample, a heterologous nucleic acid would be a nucleic acid encoding adystrophin polypeptide, or a fragment thereof, for example a codonoptimized mini-dystrophin transgene described in WO 2017/221145, andincorporated herein by reference, for use in the treatment of Duchennemuscular dystrophy.

A further exemplary heterologous nucleic acid comprises a wild-typecoding sequence, or a fragment thereof (e.g., truncated, internaldeletion), of one of the following genes, and may or may not becodon-optimized:

ABCA7 COL17A1 GBA IDUA PCSK9 SGSH ABCD1 COL4A GBE1 IL2RG PDE6C SH3TC2ACAN COL4A3 GDAP1 IMPDH1 PDE6H SLC25A13 ADA COL4A4 GJB1 ITGB2 PINK1SLC25A15 ADA2 COL7A1 GLA ITGB4 PKLR SLC26A2 ADAM10 CPS1 GLB1 JAG1 PMP22SMN AGL CRB1 GLB1 KDM6A PON1 SMPD1 AIPL1 CRX GNAT2 KMT2D PPT1 SNCA APOBCTNS GNE LAMA3 PRKN SORD APOE4 CTSD GRN LAMB3 PRPF31 SPATA7 APP CTSF GRNLAMC2 PRPF8 SPINK5 ARG1 CYBA GRS LAMP2 PRPH2 TGM1 ARSA CYBB GUCA1B LCA5PSEN1 TPP1 ARSB CYP21A2 GUCY2D LDLR PSEN2 TULP1 ASL DDC GYG1 LPL PYGLUGT1A1 ASS1 DMD HBA1 LRAT PYGM VCP ATF6 DMPK HBA2 LRRK2 RD3 VEGF ATP7BDYSF HBB MFN2 RDH12 VEGFA C9orf72 F12 HEXA MPZ RHO VPS13C CEP290 F8 HEXBMTM1 RP1 VPS35 CFTR F9 HGD NAGLU RPE65 WAS CHM FANCA HGH NAGS RPGR WIPF1CHMP2B FBLN5 HGSNAT NCF1 RPGRIP1 XPNPEP2 CLN2 FGF-1 HINT1 NCF2 RS1 BAG3CLN3 FGFR2 HMBS NCF4 SCL37A4 ATP8B1 CLN5 FGFR3 HNRNPA1 NOTCH2 SCN1AABCB11 CLN6 FXN HNRNPA2B1 OAT SERPINA1 ABCB4 CNBP G6PC HTRA1 OTCSERPING1 CNGA3 GAA HTT PAH SGCA CNGB3 GALNS IDS PARK7 SGCG

As used herein, the term “homologous,” or “homology,” refers to two ormore reference entities (e.g., nucleotide or polypeptide sequences) thatshare at least partial identity over a given region or portion. Forexample, when an amino acid position in two peptides is occupied byidentical amino acids, the peptides are homologous at that position.Notably, a homologous peptide will retain activity or functionassociated with the unmodified or reference peptide and the modifiedpeptide will generally have an amino acid sequence “substantiallyhomologous” with the amino acid sequence of the unmodified sequence.When referring to a polypeptide, nucleic acid or fragment thereof,“substantial homology” or “substantial similarity,” means that whenoptimally aligned with appropriate insertions or deletions with anotherpolypeptide, nucleic acid (or its complementary strand) or fragmentthereof, there is sequence identity in at least about 95% to 99% of thesequence. The extent of homology (identity) between two sequences can beascertained using computer program or mathematical algorithm. Suchalgorithms that calculate percent sequence homology (or identity)generally account for sequence gaps and mismatches over the comparisonregion or area. Exemplary programs and algorithms are provided below.

As used herein, the terms “host cell,” “host cell line,” and “host cellculture” are used interchangeably and refer to a cell into which anexogenous nucleic acid has been introduced, and includes the progeny ofsuch a cell. A host cell includes a “transfectant,” “transformant,”“transformed cell,” and “transduced cell,” which includes the primarytransfected, transformed or transduced cell, and progeny derivedtherefrom, without regard to the number of passages. In someembodiments, a host cell is a packaging cell for production of a rAAVvector.

As used herein, the term “host cell DNA” or “HCDNA” refers to residualDNA, derived from a host cell culture which produced a rAAV vector, andpresent in a chromatography fraction (e.g., an affinity eluate, an AEXeluate, a wash) or a chromatography load (e.g., an affinity load, an AEXload). Host cell DNA may be measured by methods know in the art such asqPCR to detect a sequence unique to the host cells. General DNAconcentrations may be estimated using fluorescence dyes (e.g. PicoGreen®or SYBR® Green), absorbance measurement (e.g. at 260 nm, or 254 nm) orelectrophoretic techniques (e.g. agarose gel electrophoresis, orcapillary electrophoresis). An amount of HCDNA present in an eluate maybe expressed relative to the amount of vg present in the eluate, forexample, ng HCDNA/1×10¹⁴ vg or pg HCDNA/1×10⁹ vg. An amount of HCDNApresent in an eluate may be expressed relative to the amount of vgpresent in a volume of eluate, for example, pg HCDNA/mL eluate.

As used herein, the term “host cell protein” or “HCP” refers to residualprotein, derived from a host cell culture which produced a rAAV vector,present in a chromatography fraction (e.g., an affinity eluate, an AEXeluate, a wash) or a chromatography load (e.g., an affinity load, an AEXload). Host cell protein may be measured by methods known in the art,such as ELISA. Host cell protein can be semi-quantitatively measured byvarious electrophoretic staining methods (e.g., silver stain SDS-PAGE,SYPRO® Ruby stain SDS-PAGE, and/or Western blot). An amount of HCPpresent in an eluate may be expressed relative to the amount of vgpresent, for example, ng HCP/1×10¹⁴ vg or pg HCP/1×10⁹ vg.

As used herein, the term “identity” or “identical to” refers to theoverall relatedness between polymeric molecules, e.g., between nucleicacid molecules (e.g., DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “substantially identical” to one another if theirsequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99% or more identical.

Calculation of the percent identity of two nucleic acid or polypeptidesequences, for example, can be performed by aligning two sequences foroptimal comparison purposes (e.g., gaps can be introduced in one or bothof a first and a second sequence for optimal alignment and non-identicalsequences can be disregarded for comparison purposes). In certainembodiments, the length of a sequence aligned for comparison purposes isat least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or 100% of the length of areference sequence. Nucleotides at corresponding positions are thencompared. When a position in a first sequence is occupied by the sameresidue (e.g., nucleotide or amino acid) as the corresponding positionin a second sequence, then the molecules are identical at that position.The percent identity between two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm.

To determine percent identity, or homology, sequences can be alignedusing the methods and computer programs, including BLAST, available overthe world wide web at ncbi.nlm.nih.gov/BLAST/. Another alignmentalgorithm is FASTA, available in the Genetics Computing Group (GCG)package, from Madison, Wis., USA. Other techniques for alignment aredescribed in Methods in Enzymology, vol. 266: Computer Methods forMacromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press,Inc. Of particular interest are alignment programs that permit gaps inthe sequence. Smith-Waterman is one type of algorithm that permits gapsin sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also,the GAP program using the Needleman and Wunsch alignment method can beutilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

Also of interest is the BestFit program using the local homologyalgorithm of Smith and Waterman (1981, Advances in Applied Mathematics2: 482-489) to determine sequence identity. The gap generation penaltywill generally range from 1 to 5, usually 2 to 4 and in some embodimentswill be 3. The gap extension penalty will generally range from about0.01 to 0.20 and in some instances will be 0.10. The program has defaultparameters determined by the sequences inputted to be compared.Preferably, the sequence identity is determined using the defaultparameters determined by the program. This program is available alsofrom Genetics Computing Group (GCG) package, from Madison, WI, USA.

Another program of interest is the FastDB algorithm. FastDB is describedin Current Methods in Sequence Comparison and Analysis, MacromoleculeSequencing and Synthesis, Selected Methods and Applications, pp.127-149, 1988, Alan R. Liss, Inc. Percent sequence identity iscalculated by FastDB based upon the following parameters: MismatchPenalty: 1.00; Gap Penalty: 1.00; Gap Size Penalty: 0.33; and JoiningPenalty: 30.0.

As used herein, the term “impurity” refers to any molecule other thanthe full rAAV vector being purified that is also present in a solutioncomprising the rAAV vector being purified. Impurities include emptycapsids, intermediate capsids, biological macromolecules such as DNA,RNA, non-AAV proteins (e.g., host cell proteins), AAV aggregates,damaged AAV capsids, molecules that are part of an absorbent used forchromatography that may leach into a sample during prior purificationsteps, endotoxins, cell debris and chemicals from cell culture,including media components, plasmid DNA from transfection, anadventitious agent, bacteria and viruses.

As used herein, the terms “inverted terminal repeat,” “ITR,” “terminalrepeat,” and “TR” refer to palindromic terminal repeat sequences at ornear the ends of the AAV virus genome, comprising mostly complementary,symmetrically arranged sequences. These ITRs can fold over to formT-shaped hairpin structures that function as primers during initiationof DNA replication. They are also needed for viral genome integrationinto host genome, for the rescue from the host genome; and for theencapsidation of viral nucleic acid into mature virions. The ITRs arerequired in cis for vector genome replication and its packaging intoviral particles. “5′ ITR” refer to the ITR at the 5′ end of the AAVgenome and/or 5′ to a recombinant transgene. “3′ ITR” refers to the ITRat the 3′ end of the AAV genome and/or 3′ to a recombinant transgene.Wild-type ITRs are approximately 145 bp in length. A modified, orrecombinant ITR, may comprise a fragment or portion of a wild-type AAVITR sequence. One of ordinary skill in the art will appreciate thatduring successive rounds of DNA replication ITR sequences may swap suchthat the 5′ ITR becomes the 3′ ITR, and vice versa. In some embodiments,at least one ITR is present at the 5′ and/or 3′ end of a recombinantvector genome such that the vector genome can be packaged into a capsidto produce a rAAV vector (also referred to herein as “rAAV vectorparticle” or “rAAV viral particle”) comprising the vector genome.

The ITRs are required in cis for vector genome replication and itspackaging into viral particles. “5′ ITR” refer to the ITR at the 5′ endof the AAV genome and/or 5′ to a recombinant transgene. “3′ ITR” refersto the ITR at the 3′ end of the AAV genome and/or 3′ to a recombinanttransgene. Wild-type ITRs are approximately 145 bp in length. Amodified, or recombinant ITR, may comprise a fragment or portion of awild-type AAV ITR sequence. One of ordinary skill in the art willappreciate that during successive rounds of DNA replication ITRsequences may swap such that the 5′ ITR becomes the 3′ ITR, and viceversa.

As used herein, the term “isolated” refers to a substance or compositionthat is 1) designed, produced, prepared, and or manufactured by the handof man and/or 2) separated from at least one of the components withwhich it was associated when initially produced (whether in natureand/or in an experimental setting). Generally, isolated compositions aresubstantially free of one or more materials with which they normallyassociate with in nature, for example, one or more protein, nucleicacid, lipid, carbohydrate and/or cell membrane. The term “isolated” doesnot exclude man-made combinations, for example, a recombinant nucleicacid, a recombinant vector genome (e.g., rAAV vector genome), a rAAVvector particle (e.g., such as, but not limited to, a rAAV vectorparticle comprising an AAV9 capsid) that packages, e.g., encapsidates, avector genome and a pharmaceutical formulation. The term “isolated” alsodoes not exclude alternative physical forms of the composition, such ashybrids/chimeras, multimers/oligomers, modifications (e.g.,phosphorylation, glycosylation, lipidation), variants or derivatizedforms, or forms expressed in host cells that are man-made.

Isolated substances or compositions may be separated from about 10%,about 20%, about 30%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more thanabout 99% of the other components with which they were initiallyassociated. In some embodiments, isolated agents are about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.As used herein, a substance is “pure” if it is substantially free ofother components. In some embodiments, as will be understood by thoseskilled in the art, a substance may still be considered “isolated” oreven “pure,” after having been combined with certain other componentssuch as, for example, one or more carriers or excipients (e.g., buffer,solvent, water, etc.); in such embodiments, percent isolation or purityof the substance is calculated without including such carriers orexcipients.

As used herein, the term “load chase” refers to a solution applied to acolumn after the load or load solution (as defined, infra) has beenapplied. A load chase serves to complete application of the load or loadsolution and to remove unbound material from the column.

As used herein, the terms “load” or “load solution” refer to anymaterial (e.g., a solution) containing a product of interest (e.g., afull rAAV vector) that is loaded onto a chromatography stationary phase.In some embodiments, a “load solution” is exposed to a chromatographystationary phase. In some embodiments, a load solution is an affinityeluate. In some embodiments, a load solution is a diluted, andoptionally filtered affinity eluate.

As used herein, the terms “stationary phase” or “chromatographystationary phase” are used to refer to any substance that can be usedfor separation of a product from another substance (e.g., an impurity).In some embodiments, a chromatography stationary phase is a resin, amedia, a membrane, a membrane adsorber, or a monolith. In someembodiments, a chromatography stationary phase is a media that binds toAAV capsids under certain conditions. In some embodiments, achromatography stationary phase is an ion exchange media (e.g., an anionexchange media, a cation exchange media). In some embodiments, achromatography stationary phase is POROS™ 50 HQ.

As used herein, the term “modifier,” or “mobile phase modifier” is acomponent of the mobile phase that modifies the mobile phase in order toalter the chromatography. Such altering of the chromatography resultsin, for example, the removal, or washing off of, impurities from thestationary phase, or elution of a product or material of interest fromthe stationary phase (e.g., a rAAV vector). Examples of “modifiers”include a salt, a detergent, an amino acid (e.g., arginine, histidine,citrulline, glycine), an organic solvent (e.g., ethanol, ethyleneglycol), a chaotropic agent (e.g., urea), or a displacer (also referredto as a selective elution agent).

As used herein, the terms “nucleic acid sequence,” “nucleotidesequence,” and “polynucleotide” refer interchangeably to any moleculecomposed of or comprising monomeric nucleotides connected byphosphodiester linkages. A nucleic acid may be an oligonucleotide or apolynucleotide. Nucleic acid sequences are presented herein in thedirection from the 5′ to the 3′ direction. A nucleic acid sequence(i.e., a polynucleotide) of the present disclosure can be adeoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) moleculeand refers to all forms of a nucleic acid such as, double strandedmolecules, single stranded molecules, small or short hairpin RNA(shRNA), micro RNA, small or short interfering RNA (siRNA),trans-splicing RNA, antisense RNA, messenger RNA, transfer RNA,ribosomal RNA.

Where a polynucleotide is a DNA molecule, that molecule can be a gene, acDNA, an antisense molecule or a fragment of any of the foregoingmolecules. Nucleotides are indicated herein by a single letter code:adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) anduracil (U). A nucleotide sequence may be chemically modified orartificial. Nucleotide sequences include peptide nucleic acids (PNA),morpholinos and locked nucleic acids (LNA), as well as glycol nucleicacids (GNA) and threose nucleic acids (TNA). Each of these sequences isdistinguished from naturally-occurring DNA or RNA by changes to thebackbone of the molecule. Also, phosphorothioate nucleotides may beused. Other deoxynucleotide analogs include methylphosphonates,phosphoramidates, phosphorodithioates, N3′-P5′-phosphoramidates, andoligoribonucleotide phosphorothioates and their 2′-O-allyl analogs and2′-O-methylribonucleotide methylphosphonates which may be used in anucleotide sequence of the disclosure.

As used here, the term “nucleic acid construct,” refers to anon-naturally occurring nucleic acid molecule resulting from the use ofrecombinant DNA technology (e.g., a recombinant nucleic acid). A nucleicacid construct is a nucleic acid molecule, either single or doublestranded, which has been modified to contain segments of nucleic acidsequences, which are combined and arranged in a manner not found innature. A nucleic acid construct may be a “vector” (e.g., a plasmid, arAAV vector genome, an expression vector, etc.), that is, a nucleic acidmolecule designed to deliver exogenously created DNA into a host cell.

As used herein, the term “operably linked” refers to a linkage ofnucleic acid sequence (or polypeptide) elements in a functionalrelationship. A nucleic acid is operably linked when it is placed into afunctional relationship with another nucleic acid sequence. Forinstance, a promoter or other transcription regulatory sequence (e.g.,an enhancer) is operably linked to a coding sequence if it affects thetranscription of the coding sequence. In some embodiments, operablylinked means that nucleic acid sequences being linked are contiguous. Insome embodiments, operably linked does not mean that nucleic acidsequences are contiguously linked, rather intervening sequences arebetween those nucleic acid sequences that are linked.

As used herein, the term “percent vector genome (VG) dilution yield” or“% VG dilution yield” refers to the amount of VG present in a dilutedaffinity pool (also referred to herein as a diluted affinity eluate) asa percentage of the amount of VG present in the affinity pool (alsoreferred to herein as an affinity eluate) prior to dilution. Forinstance, % VG dilution yield=((amount of VG in diluted affinitypool)/(amount of VG in affinity pool))*100.

As used herein, the term “percent VG column yield” or “% VG columnyield” refers to the amount of vector genomes (VG) present in a pooledeluate collected from an AEX column (i.e., an AEX pool) as a percentageof the amount of VG present in an affinity eluate that has been dilutedonly, or diluted and filtered.

In some embodiments, an affinity eluate comprising a rAAV vector to bepurified has been diluted only and is referred to as a “diluted affinitypool.” Optionally, the rAAV vector to be purified is harvested from a250 L or 2000 L vessel (e.g., a single use bioreactor (SUB)). Forinstance, % VG column yield=((amount of VG in AEX pool)/(amount of VG indiluted affinity pool))*100.

In some embodiments, an affinity eluate comprising a rAAV vector to bepurified has been diluted and filtered and is referred to as an “AEXload.” Optionally, the rAAV vector to be purified is harvested from asmall scale (e.g., less than 250 L) vessel (e.g., bioreactor). Forinstance, % VG column yield=((amount of VG in AEX pool)/(amount of VG indiluted and filtered affinity pool))*100.

As used herein, the term “percent VG step yield” or “% VG step yield”refers to the amount of VG in a pooled eluate collected from an AEXcolumn (i.e., an AEX pool) as a percentage of the amount of VG presentin the affinity pool (also referred to herein as an affinity eluate)prior to dilution or filtration. For instance, % VG step yield=((amountof VG in AEX pool)/(amount of VG in affinity pool))*100.

As used herein, the term “pharmaceutically acceptable” and“physiologically acceptable” refers to a biologically acceptableformulation, gaseous, liquid or solid, or mixture thereof, which issuitable for one or more routes of administration, in vivo delivery orcontact.

As used herein, the terms “polypeptide,” “protein,” “peptide” or“encoded by a nucleic acid sequence” (i.e., encode by a polynucleotidesequence, encoded by a nucleotide sequence) refer to full-length nativesequences, as with naturally occurring proteins, as well as functionalsubsequences, modified forms or sequence variants so long as thesubsequence, modified form or variant retains some degree offunctionality of the native full-length protein. In methods and uses ofthe disclosure, such polypeptides, proteins and peptides encoded by thenucleic acid sequences can be but are not required to be identical tothe endogenous protein that is defective, or whose expression isinsufficient, or deficient in a subject treated with gene therapy.

As used herein, the term “recombinant,” refers to a vector,polynucleotide (e.g., a recombinant nucleic acid), polypeptide or cellthat is the product of various combinations of cloning, restriction orligation steps (e.g., relating to a polynucleotide or polypeptidecomprised therein), and/or other procedure that results in a constructthat is distinct from a product found in nature. A recombinant virus orvector (e.g., rAAV vector) comprises a vector genome comprising arecombinant nucleic acid (e.g., a nucleic acid comprising a transgeneand one or more regulatory elements). The terms respectively includereplicates of the original polynucleotide construct and progeny of theoriginal virus construct.

As used herein, the term “step elution” refers to application of asolution with a defined pH, conductivity, and/or modifier concentrationto a chromatography stationary phase (including e.g., monolith, media,resin, membrane). A series of step elutions (e.g., with increasingconductivity or salt concentration) can be conducted to optimizeseparations. Each step elution solution has a defined composition thatdoes not change during its application. Over the course of the stepelution, as the series of solutions (e.g., a load chase, a pHstabilization solution, a wash buffer, an elution buffer) are applied tothe stationary phase, the pH, conductivity and/or modifier concentrationis increased, or decreased, relative to a preceding solution in theseries. For example, at the start of a step elution series, theconcentration of a modifier (e.g., a salt, e.g., sodium acetate) in thefirst solution is low, e.g., 0 to 10 mM, e.g., about 1 mM, about 2 mM,about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM,about 9 mM, about 10 mM). In each subsequent solution in the series, theconcentration of the salt is increased, such that over the course of 2to 20 solutions, the concentration of the salt is increased to, forexample, 50 mM to 300 mM (e.g., about 50 mM, about 60 mM, about 70 mM,about 80 mM, about 90 mM, about 100 mM, about 120 mM, about 140 mM,about 160 mM, about 180 mM, about 200 mM). The salt concentration in theseries of 2 to 20 (or more) solutions is not necessarily varied in equalor proportional increments.

In some embodiments, a step elution comprises 2 to 20 solutions, 2 to 10solutions, 10 to 20 solutions, for example 2, 3, 4, 5,6, 7, 8, 19, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more solutions. In someembodiments, rAAV capsids (e.g., full, intermediate, empty) are bound toa stationary phase during loading of a solution comprising the rAAVcapsid onto an AEX stationary phase. During a step elution, as a pH,conductivity and/or modifier concentration is varied, full rAAV vectorsare preferentially released (eluted) from the stationary phase, andempty capsids are preferentially retained on the stationary phase. Emptycapsids are released in greater amounts as the concentration of modifier(e.g., salt) increases. Elution of full rAAV vector from the stationaryphase can be monitored during step elution by measuring A260 and A280 ofthe eluate, such that an increase in the ratio of A260/A280 isindicative of an increase in the percentage of full rAAV vector in theeluate, and conversely, a decrease in the A260/A280 ratio is indicativeof a decrease in the percentage of full rAAV vector and an increase inthe percentage of empty capsids. In some embodiments, an absorbance ofat least one fraction of eluate is measured using a method such asanalytical size exclusion chromatography (SEC) in a high performanceliquid chromatography (HPLC) system, on-line UV trace, off-line UVmethods, etc., and wherein the absorbance is measured at one or morewavelengths (e.g., 260 nm and/or 280 nm).

As used herein, the term “subject” refers to an organism, for example, amammal (e.g., a human, a non-human mammal, a non-human primate, aprimate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, acat, a dog). In some embodiments, a human subject is an adult,adolescent, or pediatric subject. In some embodiments, a subject issuffering from a disease, disorder or condition, e.g., a disease,disorder or condition that can be treated as provided herein. In someembodiments, a subject is suffering from a disease, disorder orcondition associated with deficient or dysfunctional dystrophin, e.g.,Duchenne muscular dystrophy. In some embodiments, a subject issusceptible to a disease, disorder, or condition. In some embodiments, asusceptible subject is predisposed to and/or shows an increased risk (ascompared to the average risk observed in a reference subject orpopulation) of developing a disease, disorder or condition. In someembodiments, a subject displays one or more symptoms of a disease,disorder or condition. In some embodiments, a subject does not display aparticular symptom (e.g., clinical manifestation of disease) orcharacteristic of a disease, disorder, or condition. In someembodiments, a subject does not display any symptom or characteristic ofa disease, disorder, or condition. In some embodiments, a subject is ahuman patient. In some embodiments, a subject is an individual to whomdiagnosis and/or therapy is and/or has been administered (e.g., genetherapy for Duchenne muscular dystrophy). In some embodiments, a subjectis a human patient with Duchenne muscular dystrophy.

Disease, disorders and conditions that can be treated using a rAAVvector purified according to the methods set forth herein include, forexample a metabolic disease or disorder (e.g., Fabry disease, Gaucherdisease, phenylketonuria, glycogen storage disease); a urea cycledisease or disorder (e.g., ornithine transcarbamylase deficiency); alysosomal storage disease or disorder (e.g., metachromaticleukodystrophy, mucopolysaccharidosis); a liver disease or disorder(e.g., progressive familial intrahepatic cholestasis type 1-3); a blooddisease or disorder (Hemophilia A, Hemophilia B, a thalassemia); acancer (e.g., a carcinoma, a sarcoma, a blood cancer); a genetic diseaseor disorder (e.g., cystic fibrosis); or an infectious disease (e.g.,HIV).

Diseases, disorders and conditions that can be treated using a rAAVvector purified according to the methods set forth herein include, forexample: 21-hydroxylase-deficient congenital adrenal hyperplasia,achondrogenesis Type 1B, achondroplasia, achromatopsia, acidsphingomyelinase deficiency (Niemann-Pick disease type A or B), acuteintermittent porphyria, adenosine deaminase 2 deficiency, adenosinedeaminase deficiency (e.g., severe combined immunodeficiency, X-linked),adrenoleukodystrophy (e.g., X-linked), age-related macular degeneration(e.g., neovascular, wet), Alagille syndrome, alkaptonuria, alpha-1antitrypsin deficiency, alpha-thalassemia, Alport syndrome, Alzheimerdisease, Apert syndrome, arginase deficiency, argininosuccinate lyase(ASL) deficiency, argininosuccinate synthase (ASS1) deficiency(citrullinemia type 1), aromatic L-amino acid decarboxylase deficiency,autosomal recessive congenital ichthyosis, Becker muscular dystrophy,beta-thalassemia, carbamoylphosphatase synthetase I deficiency, ceroidlipofuscinosis, Charcot-Marie-Tooth neuropathy, choroideremia, chronicgranulomatous disease, citrin deficiency, Crigler-Najjar syndrome type 1and 2, critical limb ischemia, cystic fibrosis, cystinosis, Danondisease, diabetic macular retinopathy, dominant inherited short stature,Dravet syndrome, Duchenne muscular dystrophy, dysferlinopathy (e.g.,Miyoshi myopathy, limb-girdle muscular dystrophy 2B), dystrophicepidermolysis bullosa, Fabry disease, familial hypercholesterolemia,familial lipoprotein lipase deficiency, Fanconia anemia (e.g., Fanconiaanemia A), Friedreich's ataxia, frontotemporal dementia, Gaucherdisease, glycogen storage disease type 1 A and 1B (Von Gierke'sdisease), glycogen storage disease type III, glycogen storage diseasetype IV, glycogen storage disease type V, glycogen storage disease typeVI, glycogen storage disease type XV, GM1 gangliosidosis, gyrateatrophy, hemophilia A, hemophilia B, hereditary angiodema, types I-III,Huntington's disease, inclusion body myositis, junctional epidermolysisbullosa, Kabuki Syndrome, Leber congenital amaurosis, leukocyte adhesiondefect type 1, limb girdle muscular dystrophy, limb girdle musculardystrophy type 2C (gamma-sarcoglycanopathy), limb girdle musculardystrophy type 2D, metachromatic leukodystrophy, mucopolysaccharidosisType I, mucopolysaccharidosis type II (Hunter syndrome),mucopolysaccharidosis type IIIA, mucopolysaccharidosis type IIIB,mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID,mucopolysaccharidosis type IVA (Morquio A syndrome),mucopolysaccharidosis type IVB (Morquio B syndrome),mucopolysaccharidosis type VI (Maroteaux-Lamy), myotonic dystrophy type1, myotonic dystrophy type 2, N-acetylglutamate synthase (NAGS)deficiency, Netherton syndrome, neuronal ceroid lipofuscinosis,ornithine translocase deficiency, ornithine transcarbamylase deficiencydisease, Parkinson's disease, phenylketonuria, Pompe, progressivefamilial intrahepatic cholestasis type 1-3, progressive myofibrillarmyopathy, pyruvate kinase deficiency, retinitis pigmentosa,RPE65-related Leber congenital amaurosis, Sandhoff disease, sickle celldisease, spinal muscular atrophy, Tay Sachs disease, Wilson disease,Wiskott-Aldrich syndrome, Wiskott-Aldrich syndrome 2, X-linkedadrenoleukodystrophy, X-linked chronic granulomatous disease, X-linkedmyotubular myopathy, X-linked retinitis pigmentosa, X-linkedretinoschisis and X-linked severe combined immunodeficiency.

As used herein, the term “substantially” refers to the qualitativecondition of exhibition of total or near-total extent or degree of acharacteristic or property of interest. One of ordinary skill in the artwill understand that biological and chemical phenomena rarely, if ever,go to completion and/or proceed to completeness or achieve or anabsolute result. The term “substantially” is therefore used herein tocapture the potential lack of completeness inherent in many biologicaland chemical phenomena.

As used herein, the term “therapeutic polypeptide” is a peptide,polypeptide or protein (e.g., enzyme, structural protein, transmembraneprotein, transport protein) that may alleviate or reduce symptoms thatresult from an absence or defect in a protein in a target cell (e.g., anisolated cell) or organism (e.g., a subject). A therapeutic polypeptideor protein encoded by a transgene is one that confers a benefit to asubject, e.g., to correct a genetic defect, to correct a deficiency in agene related to expression or function. Similarly, a “therapeutictransgene” is the transgene that encodes the therapeutic polypeptide. Insome embodiments, a therapeutic polypeptide, expressed in a host cell,is an enzyme expressed from a transgene (i.e., an exogenous nucleic acidthat has been introduced into the host cell). In some embodiments, atherapeutic polypeptide is a dystrophin protein, or fragment thereof,expressed from a therapeutic transgene transduced into a muscle cell(e.g., a skeletal muscle cell).

As used herein, the term “therapeutically effective amount” refers to anamount that produces the desired therapeutic effect for which it isadministered. In some embodiments, the term refers to an amount that issufficient, when administered to a population suffering from orsusceptible to a disease, disorder or condition in accordance with atherapeutic dosing regimen, to treat the disease, disorder or condition.In some embodiments, a therapeutically effective amount is one thatreduces the incidence and/or severity of, and/or delays onset of, one ormore symptoms of the disease, disorder, and/or condition. Those ofordinary skill in the art will appreciate that the term “therapeuticallyeffective amount” does not in fact require successful treatment beachieved in a particular individual. Rather, a therapeutically effectiveamount may be that amount that provides a particular desiredpharmacological response in a significant number of subjects whenadministered to patients in need of such treatment.

As used herein, the term “transgene” is used to mean any heterologouspolynucleotide for delivery to and/or expression in a host cell, targetcell or organism (e.g., a subject). Such “transgene” may be delivered toa host cell, target cell or organism using a vector (e.g., rAAV vector).A transgene may be operably linked to a control sequence, such as apromoter. It will be appreciated by those of skill in the art thatexpression control sequences can be selected based on an ability topromote expression of the transgene in a host cell, target cell ororganism. Generally, a transgene may be operably linked to an endogenouspromoter associated with the transgene in nature, but more typically,the transgene is operably linked to a promoter with which the transgeneis not associated in nature. An example of a transgene is a nucleic acidencoding a therapeutic polypeptide, for example an dystrophinpolypeptide or fragment thereof, and an exemplary promoter is one notoperable linked to a nucleotide encoding dystrophin in nature. Such anon-endogenous promoter can include a CBh promoter or a muscle specificpromoter, among many others known in the art.

A nucleic acid of interest can be introduced into a host cell by a widevariety of techniques that are well-known in the art, includingtransfection and transduction.

“Transfection” is generally known as a technique for introducing anexogenous nucleic acid into a cell without the use of a viral vector. Asused herein, the term “transfection” refers to transfer of a recombinantnucleic acid (e.g., an expression plasmid) into a cell (e.g., a hostcell) without use of a viral vector. A cell into which a recombinantnucleic acid has been introduced is referred to as a “transfected cell.”A transfected cell may be a host cell (e.g., a CHO cell, Prol10 cell,HEK293 cell) comprising an expression plasmid/vector for producing arecombinant AAV vector. In some embodiments, a transfected cell (e.g., apacking cell) may comprise a plasmid comprising a transgene (e.g., andystrophin transgene), a plasmid comprising an AAV rep gene and an AAVcap gene and a plasmid comprising a helper gene. Many transfectiontechniques are known in the art, which include, but are not limited to,electroporation, calcium phosphate precipitation, microinjection,cationic or anionic liposomes, and liposomes in combination with anuclear localization signal.

As used herein, the term “transduction” refers to transfer of a nucleicacid (e.g., a vector genome) by a viral vector (e.g., rAAV vector) to acell (e.g., a target cell, e.g., a muscle cell). In some embodiments, agene therapy for Duchenne muscular dystrophy includes transducing avector genome comprising a modified nucleic acid encoding dystrophin, ora fragment thereof, into a muscle cell. A cell into which a transgenehas been introduced by a virus or a viral vector is referred to as a“transduced cell.” In some embodiments, a transduced cell is an isolatedcell and transduction occurs ex vivo. In some embodiments, a transducedcell is a cell within an organism (e.g., a subject) and transductionoccurs in vivo. A transduced cell may be a target cell of an organismwhich has been transduced by a recombinant AAV vector such that thetarget cell of the organism expresses a polynucleotide (e.g., atransgene, e.g., a modified nucleic acid encoding dystrophin, or afragment thereof).

Cells that may be transduced include a cell of any tissue or organ type,or any origin (e.g., mesoderm, ectoderm or endoderm). Non-limitingexamples of cells include liver (e.g., hepatocytes, sinusoidalendothelial cells), pancreas (e.g., beta islet cells, exocrine), lung,central or peripheral nervous system, such as brain (e.g., neural orependymal cells, oligodendrocytes) or spine, kidney, eye (e.g.,retinal), spleen, skin, thymus, testes, lung, diaphragm, heart(cardiac), muscle or psoas, or gut (e.g., endocrine), adipose tissue(white, brown or beige), muscle (e.g., fibroblasts, myocytes),synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelialcells, salivary gland cells, inner ear nervous cells or hematopoietic(e.g., blood or lymph) cells. Additional examples include stem cells,such as pluripotent or multipotent progenitor cells that develop ordifferentiate into liver (e.g., hepatocytes, sinusoidal endothelialcells), pancreas (e.g., beta islet cells, exocrine cells), lung, centralor peripheral nervous system, such as brain (e.g., neural or ependymalcells, oligodendrocytes) or spine, kidney, eye (e.g., retinal), spleen,skin, thymus, testes, lung, diaphragm, heart (cardiac), muscle or psoas,or gut (e.g., endocrine), adipose tissue (white, brown or beige), muscle(e.g., fibroblast, myocytes), synoviocytes, chondrocytes, osteoclasts,epithelial cells, endothelial cells, salivary gland cells, inner earnervous cells or hematopoietic (e.g., blood or lymph) cells.

In some embodiments, particular areas of a tissue or organ (e.g.,muscle) may be transduced by a rAAV vector (e.g., a rAAV vector with adystrophin, or portion of dystrophin, transgene) that is administered tothe tissue or organ. In some embodiments, a muscle cell is transducedwith a rAAV comprising a dystrophin transgene. In some embodiments, askeletal muscle cell is transduced with a rAAV comprising a dystrophintransgene. In some embodiments, a cardiac muscle cell is transduced witha rAAV comprising a dystrophin transgene.

As used herein, the term “vector” refers to a plasmid, virus (e.g., arAAV), cosmid, or other vehicle that can be manipulated by insertion orincorporation of a nucleic acid (e.g., a recombinant nucleic acid). Avector can be used for various purposes including, e.g., geneticmanipulation (e.g., cloning vector), to introduce/transfer a nucleicacid into a cell, to transcribe or translate an inserted nucleic acid ina cell. In some embodiments a vector nucleic acid sequence contains atleast an origin of replication for propagation in a cell. In someembodiments, a vector nucleic acid includes a heterologous nucleic acidsequence, an expression control element(s) (e.g., promoter, enhancer), aselectable marker (e.g., antibiotic resistance), a poly-adenosine(polyA) sequence and/or an ITR. In some embodiments, when delivered to ahost cell, the nucleic acid sequence is propagated. In some embodiments,when delivered to a host cell, either in vitro or in vivo, the cellexpresses the polypeptide encoded by the heterologous nucleic acidsequence. In some embodiments, when delivered to a host cell, thenucleic acid sequence, or a portion of the nucleic acid sequence ispackaged into a capsid. A host cell may be an isolated cell or a cellwithin a host organism. In addition to a nucleic acid sequence (e.g.,transgene) which encodes a polypeptide or protein, additional sequences(e.g., regulatory sequences) may be present within the same vector(i.e., in cis to the gene) and flank the gene. In some embodiments,regulatory sequences may be present on a separate (e.g., a second)vector which acts in trans to regulate the expression of the gene.Plasmid vectors may be referred to herein as “expression vectors.”

As used herein, the term “vector genome” refers to a nucleic acid thatis packaged/encapsidated in an AAV capsid to form a rAAV vector.Typically, a vector genome includes a heterologous polynucleotidesequence (e.g., a transgene, regulatory elements, etc.) and at least oneITR. In cases where a recombinant plasmid is used to construct ormanufacture a recombinant vector (e.g., rAAV vector), the vector genomedoes not include the entire plasmid but rather only the sequenceintended for delivery by the viral vector. This non-vector genomeportion of the recombinant plasmid is referred to as the “plasmidbackbone,” which is important for cloning. selection and amplificationof the plasmid, a process that is needed for propagation of recombinantviral vector production, but which is not itself packaged orencapsidated into a rAAV vector. Typically, the heterologous sequence tobe packaged into the capsid is flanked by the ITRs such that whencleaved from the plasmid backbone, it is packaged into the capsid.

As used herein, the term “viral vector” generally refers to a viralparticle that functions as a nucleic acid delivery vehicle and whichcomprises a vector genome (e.g., comprising a transgene which hasreplaced the wild type rep and cap) packaged within the viral particle(i.e., capsid) and includes, for example, lenti- and parvo-viruses,including AAV serotypes and variants (e.g., rAAV vectors). As notedelsewhere herein, a recombinant viral vector does not comprise a virusgenome with a rep and/or a cap gene; rather, these sequences have beenremoved to provide capacity for the vector genome to carry a transgeneof interest.

The present disclosure provides methods for purification of rAAV vectors(e.g., full rAAV vectors) from host cell harvests. In particular, thedisclosure provides methods for purification of rAAV vectors (e.g., fullrAAV vectors) from other nucleic acids and proteins (including emptycapsids) produced by the host cell. Furthermore, the disclosure providesmethods for the separation of empty capsids from full rAAV vectors(e.g., rAAV vectors comprising a vector genome). Each of these aspectsof the disclosure is discussed further in the ensuing sections.

2. AAV and rAAV Vectors

AAV

As discussed supra, “adeno-associated virus” and/or “AAV” refer toparvoviruses with a linear single-stranded DNA genome and variantsthereof. The term covers all subtypes and both naturally occurring andrecombinant forms, except where required otherwise. Parvoviruses,including AAV, are useful as gene therapy vectors as they can penetratea cell and introduce a nucleic acid (e.g., transgene) into the nucleus.In some embodiments, the introduced nucleic acid (e.g., rAAV vectorgenome) forms circular concatemers that persist as episomes in thenucleus of transduced cells. In some embodiments, a transgene isinserted in specific sites in the host cell genome, for example at asite on human chromosome 19. Site-specific integration, as opposed torandom integration, is believed to likely result in a predictablelong-term expression profile. The insertion site of AAV into the humangenome is referred to as AAVS1. Once introduced into a cell,polypeptides encoded by the nucleic acid can be expressed by the cell.Because AAV is not associated with any pathogenic disease in humans, anucleic acid delivered by AAV can be used to express a therapeuticpolypeptide for the treatment of a disease, disorder and/or condition ina human subject.

Multiple serotypes of AAV exist in nature with at least fifteen wildtype serotypes having been identified from humans thus far (i.e.,AAV1-AAV15). Naturally occurring and variant serotypes are distinguishedby having a protein capsid that is serologically distinct from other AAVserotypes. AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3)including AAV type 3A (AAV3A) and AAV type 3B (AAV3B), AAV type 4(AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAVtype 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 12(AAV12), AAVrh10, AAVrh74 (see WO 2016/210170), avian AAV, bovine AAV,canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, andrecombinantly produced variants (e.g., capsid variants with insertions,deletions and substitutions, etc.), such as variants referred to as AAVtype 2i8 (AAV2i8), NP4, NP22, NP66, AAVDJ, AAVDJ/8, AAVDJ/9, AAVLK03,RHM4-1, among many others. AAV variants isolated from human CD34+ cellinclude AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7,AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14 andAAVHSC15 (Smith et al. (2014) Molecular Therapy 22(9):1625-1634).

“Primate AAV” refers to AAV that infect primates, “non-primate AAV”refers to AAV that infect non-primate mammals, “bovine AAV” refers toAAV that infect bovine mammals, and so on. Serotype distinctiveness isdetermined on the basis of the lack of cross-reactivity betweenantibodies to one AAV as compared to another AAV. Such cross-reactivitydifferences are usually due to differences in capsid protein sequencesand antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequencedifferences of AAV serotypes). However, some naturally occurring AAV orman-made AAV mutants (e.g., recombinant AAV) may not exhibit serologicaldifference with any of the currently known serotypes. These viruses maythen be considered a subgroup of the corresponding type, or more simplya variant AAV. Thus, as used herein, the term “serotype” refers to bothserologically distinct viruses, e.g., AAV, as well as viruses, e.g.,AAV, that are not serologically distinct but that may be within asubgroup or a variant of a given serotype.

A comprehensive list and alignment of amino acid sequences of capsids ofknown AAV serotypes is provided by Marsic et al. (2014) MolecularTherapy 22(11):1900-1909, especially at supplementary FIG. 1 .

Genomic sequences of various serotypes of AAV, as well as sequences ofthe native terminal repeats (ITRs), rep proteins, and capsid subunitsare known in the art. Such sequences may be found in the literature orin public databases such as GenBank. See, e.g., GenBank AccessionNumbers NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303(AAV2), NC_001729 (AAV3), AF028705.1 (AAV3B), NC_001829 (AAV4), U89790(AAV4), NC_006152 (AAV5), AF028704 (AAV6), AF513851 (AAV7), AF513852(AAV8), NC_006261 (AAV8), AY530579 (AAV9), AY631965 (AAV10), AY631966(AAV11), and DQ813647 (AAV12); the disclosures of which are incorporatedby reference herein. See also, e.g., Srivistava et al. (1983) J.Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini etal. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996)Virology 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al.(2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology33:375-383; international patent publications WO 00/28061, WO 99/61601,WO 98/11244; WO 2013/063379; WO 2014/194132; WO 2015/121501, and U.S.Pat. Nos. 6,156,303 and 7,906,111. For illustrative purposes only, wildtype AAV2 comprises a small (20-25 nm) icosahedral virus capsid of AAVcomposed of three proteins (VP1, VP2, and VP3; a total of 60 capsidproteins compose the AAV capsid) with overlapping sequences. Theproteins VP1 (735 aa; Genbank Accession No. AAC03780), VP2 (598 aa;Genbank Accession No. AAC03778) and VP3 (533 aa; Genbank Accession No.AAC03779) exist in about a 1:1:10 ratio in the capsid. That is, forAAVs, VP1 is the full length protein and VP2 and VP3 are progressivelyshorter versions of VP1, with increasing truncation of the N-terminusrelative to VP1. In one embodiment, of the method disclosed herein, arAAV vector comprises an AAV9 VP1 comprising the amino acid sequence ofSEQ ID NO:11.

Recombinant AAV (rAAV)

As discussed supra, a “recombinant adeno-associated virus” or “rAAV” isdistinguished from a wild-type AAV by replacement of all or part of theviral genome with a non-native sequence. Incorporation of a non-nativesequence within the virus defines the viral vector as a “recombinant”vector, and hence a “rAAV vector.” A rAAV vector can include aheterologous polynucleotide (e.g., human codon-optimized gene encodinghuman mini-dystrophin, e.g., SEQ ID NO:1) encoding a desired protein orpolypeptide (e.g., a dystrophin polypeptide, or fragment thereof, e.g.,SEQ ID NO:2). A recombinant vector sequence may be encapsidated orpackaged into an AAV capsid and referred to as an “rAAV vector,” an“rAAV vector particle,” “rAAV viral particle” or simply a “rAAV.”

The present disclosure provides for methods of purifying a rAAV vectorcomprising a polynucleotide sequence not of AAV origin (e.g., apolynucleotide heterologous to AAV). The heterologous polynucleotide maybe flanked by at least one, and sometimes by two, AAV terminal repeatsequences (e.g., inverted terminal repeats (ITRs)). The heterologouspolynucleotide flanked by ITRs, also referred to herein as a “vectorgenome,” typically encodes a polypeptide of interest, or a gene ofinterest (“GOI”), such as a target for therapeutic treatment (e.g., anucleic acid encoding dystrophin, or a fragment thereof, for thetreatment of Duchenne muscular dystrophy). Delivery or administration ofa rAAV vector to a subject (e.g. a patient) provides encoded proteinsand peptides to the subject. Thus, a rAAV vector can be used totransfer/deliver a heterologous polynucleotide for expression for, e.g.,treating a variety of diseases, disorders and conditions.

rAAV vector genomes generally retain 145 base ITRs in cis to theheterologous nucleic acid sequence that replaces the viral rep and capgenes. Such ITRs are necessary to produce a recombinant AAV vector;however, modified AAV ITRs and non-AAV terminal repeats includingpartially or completely synthetic sequences can also serve this purpose.ITRs form hairpin structures and function to, for example, serve asprimers for host-cell-mediated synthesis of the complementary DNA strandafter infection. ITRs also play a role in viral packaging, integration,etc. ITRs are the only AAV viral elements which are required in cis forAAV genome replication and packaging into rAAV vectors. A rAAV vectorgenome optionally comprises two ITRs which are generally at the 5′ and3′ ends of the vector genome comprising a heterologous sequence (e.g., atransgene encoding a gene of interest, or a nucleic acid sequence ofinterest including, but not limited to, an antisense, and siRNA, aCRISPR molecule, among many others). A 5′ and a 3′ ITR may both comprisethe same sequence, or each may comprise a different sequence. An AAV ITRmay be from any AAV including by not limited to serotypes 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or 11 or any other AAV. An ITR is a sequence whichmediates AAV genome replication and packaging.

A rAAV vector of the disclosure may comprise an ITR from an AAV serotype(e.g., wild-type AAV2, a fragment or variant thereof) that differs fromthe serotype of the capsid (e.g., AAV9 or other). Such a rAAV vectorcomprising at least one ITR from one serotype, but comprising an AAVcapsid protein from a different serotype, may be referred to as a hybridviral vector (see U.S. Pat. No. 7,172,893). An AAV ITR may include theentire wild type ITR sequence, or be a variant, fragment, ormodification thereof, but will retain functionality.

In some embodiments, a heterologous polypeptide comprises an ITR (e.g.,an ITR from AAV2, but can comprise an ITR from any wild type AAVserotype, or a variant thereof) positioned at the left and right ends(i.e., 5′ and 3′ termini, respectively) of a vector genome. In someembodiments, a left (e.g., 5′) ITR comprises or consists of the nucleicacid sequence of SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, a left(e.g., 5′) ITR comprises a nucleic acid sequence that is about 80%,about 85%, about 90%, about 95%, about 98%, about 99% or 100% identicalto SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, a right (e.g., 3′)ITR comprises or consists of a nucleic acid sequence of SEQ ID NO:7 orSEQ ID NO:8. In some embodiments, a right (e.g., 3′) ITR comprises anucleic acid sequence that is about 80%, about 85%, about 90%, about95%, about 98%, about 99% or 100% identical to SEQ ID NO:7 or SEQ IDNO:8. Each ITR is in cis with but may be separated from each other, orother elements in the vector genome, by a nucleic acid sequence ofvariable length, such as a recombinant nucleic acid comprising amodified nucleic acid encoding dystrophin, or a fragment thereof, andregulatory elements. In some embodiments, ITRs are AAV2 ITRs, orvariants thereof, and flank a dystrophin transgene. In some embodiments,a rAAV comprises a dystrophin transgene (e.g., comprising the nucleicacid sequence of SEQ ID NO:1) flanked by AAV2 ITRs (e.g., ITRs havingthe sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8).

In some embodiments, a rAAV vector genome is linear, single-stranded andflanked by AAV ITRs. Prior to transcription and translation of theheterologous gene, a single stranded DNA genome of approximately 4700nucleotides must be converted to a double-stranded form by DNApolymerases (e.g., DNA polymerases within the transduced cell) using thefree 3′-OH of one of the self-priming ITRs to initiate second-strandsynthesis. In some embodiments, full length-single stranded vectorgenomes (i.e., sense and anti-sense) anneal to generate a fulllength-double stranded vector genome. This may occur when multiple rAAVvectors carrying genomes of opposite polarity (i.e., sense oranti-sense) simultaneously transduce the same cell. Regardless of howthey are produced, once double-stranded vector genomes are formed, thecell can transcribe and translate the double-stranded DNA and expressthe heterologous gene.

The efficiency of transgene expression from a rAAV vector can behindered by the need to convert a single stranded rAAV genome (ssAAV)into double-stranded DNA prior to expression. This step is circumventedby using a self-complementary AAV genome (scAAV) that can package aninverted repeat genome that can fold into double-stranded DNA withoutthe need for DNA synthesis or base-pairing between multiple vectorgenomes (McCarty, (2008) Molec. Therapy 16(10):1648-1656; McCarty etal., (2001) Gene Therapy 8:1248-1254; McCarty et al., (2003) GeneTherapy 10:2112-2118). A limitation of a scAAV vector is that size ofthe unique transgene, regulatory elements and IRTs to be package in thecapsid is about half the size (i.e., ˜2,500 nucleotides of which 2,200nucleotides may be a transgene and regulatory elements, plus two copiesof the ˜145 nucleotide ITRs) of a ssAAV vector genome (i.e., ˜4,900nucleotides including two ITRs).

scAAV vector genomes are made by deleting the terminal resolution site(TRS) from one rAAV ITR of the expression plasmid, thereby preventinginitiation of replication from that end (see U.S. Pat. No. 8,784,799).AAV replication within a host cell is initiated at the wild type ITR ofthe genome and continues through the mutant ITR without terminalresolution and then back across the genome to create a dimer. The dimeris a self-complementary genome with a mutant ITR in the middle, andwild-type ITRs at each end. In some embodiments, a mutant ITR with adeleted TRS is at the 5′ end of the vector genome. In some embodiments,a mutant ITR with a deleted TRS is at the 3′ end of the vector genome.In some embodiments, a mutant ITR comprises the nucleic acid sequence ofSEQ ID NO:13 or SEQ ID NO:14.

Without wishing to be bound by theory, while the two halves of a scAAVgenome are complementary, it is unlikely that there is substantial basepairing within the capsid as many of the bases are in contact with aminoacid residues of the inner capsid shell and the phosphate backbone issequestered toward the center (McCarty, Molec. Therapy (2008)16(10):1648-1656). It likely that upon uncoating, the two halves of thescAAV genome anneal to form a dsDNA hairpin molecule, with a covalentlyclosed ITR at one end and two open-ended ITRs on the other. The ITRsflank a double-stranded region encoding, among other things, thetransgene, and regulatory elements in cis thereto.

A viral capsid of a rAAV vector may be from a wild type AAV or a variantAAV such as AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAVrh10, AAVrh74 (see WO2016/210170), AAV12, AAV2i8,AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO:5 of WO2015/013313), RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAVhu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV2i8, AAV29G,AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV avian AAV,bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, snakeAAV, goat AAV, shrimp AAV, ovine AAV and variants thereof (see, e.g.,Fields et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-RavenPublishers). Capsids may be derived from a number of AAV serotypesdisclosed in U.S. Pat. No. 7,906,111; Gao et al. (2004) J. Virol.78:6381; Morris et al. (2004) Virol. 33:375; WO 2013/063379; WO2014/194132; and include true type AAV (AAV-TT) variants disclosed in WO2015/121501, and RHM4-1, RHM15-1 through RHM15-6, and variants thereof,disclosed in WO 2015/013313. Capsids may also be derived from AAVvariants isolated from human CD34+ cell include AAVHSC1, AAVHSC2,AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10,AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15 (Smith et al. (2014)Molecular Therapy 22(9):1625-1634). One skilled in the art would knowthere are likely other AAV variants not yet identified that perform thesame or similar function. A full complement of AAV cap proteins includesVP1, VP2, and VP3. The ORF comprising nucleotide sequences encoding AAVVP capsid proteins may comprise less than a full complement AAV Capproteins or the full complement of AAV cap proteins may be provided.

In another embodiment, the present disclosure provides for the use ofancestral AAV vectors for use in therapeutic in vivo gene therapy.Specifically, in silico-derived sequences may be synthesized de novo andcharacterized for biological activities. Prediction and synthesis ofancestral sequences, in addition to assembly into a rAAV vector, may beaccomplished using methods described in WO 2015/054653, the contents ofwhich are incorporated by reference herein. Notably, rAAV vectorsassembled from ancestral viral sequences may exhibit reducedsusceptibility to pre-existing immunity in human populations as comparedto contemporary viruses or portions thereof.

In some embodiments, a rAAV vector comprising a capsid protein encodedby a nucleotide sequence derived from more than one AAV serotype (e.g.,wild type AAV serotypes, variant AAV serotypes) is referred to as a“chimeric vector” or “chimeric capsid” (See U.S. Pat. No. 6,491,907, theentire disclosure of which is incorporated herein by reference). In someembodiments, a chimeric capsid protein is encoded by a nucleic acidsequence derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more AAV serotypes.In some embodiments, a recombinant AAV vector includes a capsid sequencederived from e.g., AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrh10, AAV2i8, or variantthereof, resulting in a chimeric capsid protein comprising a combinationof amino acids from any of the foregoing AAV serotypes (see, Rabinowitzet al. (2002) J. Virology 76(2):791-801). Alternatively, a chimericcapsid can comprise a mixture of a VP1 from one serotype, a VP2 from adifferent serotype, a VP3 from yet a different serotype, and acombination thereof. For example, a chimeric virus capsid may include anAAV1 cap protein or subunit and at least one AAV2 cap protein orsubunit. A chimeric capsid can, for example include an AAV capsid withone or more B19 cap subunits, e.g., an AAV cap protein or subunit can bereplaced by a B19 cap protein or subunit. For example, in oneembodiment, a VP3 subunit of an AAV capsid can be replaced by a VP2subunit of B19.

In some embodiments, chimeric vectors have been engineered to exhibitaltered tropism or tropism for a particular tissue or cell type. Theterm “tropism” refers to preferential entry of the virus into certaincell or tissue types and/or preferential interaction with the cellsurface that facilitates entry into certain cell or tissue types. AAVtropism is generally determined by the specific interaction betweendistinct viral capsid proteins and their cognate cellular receptors(Lykken et al. (2018) J. Neurodev. Disord. 10:16). Preferably, once avirus or viral vector has entered a cell, sequences (e.g., heterologoussequences such as a transgene) carried by the vector genome (e.g., arAAV vector genome) are expressed.

A “tropism profile” refers to a pattern of transduction of one or moretarget cells, tissues and/or organs. For example, an AAV capsid may havea tropism profile characterized by efficient transduction of musclecells with only low transduction of, for example, brain cells.

3. Recombinant Nucleic Acids

Methods of the present disclosure include purification of a rAAV vectorcomprising a recombinant nucleic acid including modified nucleic acidsas well as plasmids and vector genomes that comprise a modified nucleicacid. A recombinant nucleic acid, plasmid or vector genome may compriseregulatory sequences to modulate propagation (e.g., of a plasmid) and/orcontrol expression of a modified nucleic acid (e.g., a transgene).Recombinant nucleic acids may also be provided as a component of a viralvector (e.g., a rAAV vector). Generally, a viral vector includes avector genome comprising a recombinant nucleic acid packaged in acapsid.

Modified Nucleic Acids

A modified, or variant form, of a gene, nucleic acid or polynucleotide(e.g., a transgene) refers to a nucleic acid that deviates from areference sequence. A reference sequence may be a naturally occurring,wild type sequence (e.g., a gene) and may include naturally occurringvariants (e.g., splice variants, alternative reading frames). Thoseskilled in the art will be aware that reference sequences can be foundin publicly available databases such as GenBank(ncbi.nlm.nih.gov/genbank). Modified/variant nucleic acids may havesubstantially the same, greater or lesser activity, function orexpression as compared to a reference sequence. Preferably, a modified,or variant nucleic acid, as used interchangeably herein, exhibitsimproved protein expression, e.g., a protein encoded thereby isexpressed at a detectably greater level in a cell compared with thelevel of expression of a protein provided by an endogenous gene (e.g., awild type gene, a mutant gene) in an otherwise identical cell. In someembodiments, a modified, or variant nucleic acid, as usedinterchangeably herein, exhibits improved protein expression, e.g., aprotein encoded thereby is expressed at a detectably greater level in acell compared with the level of expression of a protein provided by anendogenous gene comprising a mutation in an otherwise identical cell.

Modifications to nucleic acids include one or more nucleotidesubstitutions (e.g., substitution of 1-3, 3-5, 5-10, 10-15, 15-20,20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides), additions(e.g., insertion of 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40,40-50, 50-100 or more nucleotides), deletions (e.g., deletion of 1-3,3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or morenucleotides, deletion of a motif, domain, fragment, etc.) of a referencesequence. A modified nucleic acid may be about 50%, about 60%, about70%, about 80%, about 85%, about 90%, about 92%, about 93%, about 94%,about 95%, about 96% about 97% about 98% or about 99% identical to areference sequence.

A modified nucleic acid may encode a polypeptide with about 50%, about60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 98%,about 99% or 100% identity to a reference polypeptide. In someembodiments, a modified nucleic acid encodes a polypeptide with 100%identify to a reference polypeptide.

In some embodiments, a modified nucleic acid (e.g., transgene) encodes awild-type protein. Such modified nucleic acid may be codon optimized.“Optimized” or “codon-optimized,” as referred to interchangeably herein,refers to a coding sequence that has been optimized relative to a wildtype coding sequence or reference sequence (e.g., a coding sequence fora mini-dystrophin polypeptide, e.g., SEQ ID NO:2, a coding sequence fora deleted copper transporting ATPase polypeptide, e.g., SEQ ID NO:15) toincrease expression of the polypeptide, e.g., by minimizing usage ofrare codons, decreasing the number of CpG dinucleotides, removingcryptic splice donor or acceptor sites, removing Kozak sequences,removing ribosomal entry sites, and the like. In some embodiments, alevel of expression of a protein from a codon-optimized sequence isincreased as compared to a level of expression of a protein from a wildtype gene in an otherwise identical cell. In some embodiments, a levelof expression of a protein from a codon-optimized sequence is notincreased (e.g., expression is substantially similar) as compared to alevel of expression of a protein from a wild-type gene in an otherwiseidentical cell. In some embodiments, a level of expression of a proteinfrom a codon-optimized sequence is increased as compared to a level ofexpression of a protein from a mutant gene in an otherwise identicalcell.

Examples of modifications include elimination of one or more cis-actingmotifs and introduction of one or more Kozak sequences. In someembodiments, one or more cis-acting motifs are eliminated and one ormore Kozak sequences are introduced.

Examples of cis-acting motifs that may be eliminated include internalTATA-boxes; chi-sites; ribosomal entry sites; ARE, INS, and/or CRSsequence elements; repeat sequences and/or RNA secondary structures;(cryptic) splice donor and/or acceptor sites, branch points; andrestriction sites.

In some embodiments, a modified nucleic acid encodes a modified orvariant polypeptide. A modified polypeptide (e.g., a codon optimizedmini-dystrophin) encoded by a modified nucleic acid may retain all or apart of the function or activity of a polypeptide encoded by a wild typecoding or reference sequence. In some embodiments, a modifiedpolypeptide has one or more non-conservative or conservative amino acidchanges. In some embodiments, certain domains that have beendemonstrated to play a limited or no role in a function of a polypeptideare not present in a modified polypeptide (e.g., certain bindingdomains) (e.g., WO 2016/097219). Modified nucleic acids present in rAAVvectors may comprise fewer nucleotides than the wild type coding, orreference sequence, due to the packaging capacity of a rAAV capsid(e.g., shortened minidystrophin transgene, see WO 2001/83695; a B-domaindeleted human Factor VIII transgene, see WO 2017/074526 all of which areincorporated herein by reference), and also include shortened transgenesthat are both truncated and codon-optimized (e.g., a codon optimizedmini-dystrophin transgene described in WO 2017/221145; deleted coppertransporting ATPase2 with deletion of metal binding sites (MBS) 1-4, seeWO 2016/097219 and WO 2016/097218 all of which are incorporated hereinby reference). In some embodiments, a polypeptide encoded by a modifiednucleic acid has less than, the same, or greater, but at least a partof, a function or activity of a polypeptide encoded by a referencesequence.

Modified nucleic acids may have a modified GC content (e.g., the numberof G and C nucleotides present in a nucleic acid sequence), a modified(e.g., increased or decreased) CpG dinucleotide content and/or amodified (e.g., increased or decreased) codon adaptation index (CAI)relative to a reference and/or wild-type sequence. See, e.g., WO2017/077451 (discussing various considerations well-known in the art forcodon-optimization of nucleic acid sequences of interest, includingpublicly available software for analyzing nucleic acid sequences foroptimization). As used herein, modified refers to a decrease or anincrease in a particular value, amount or effect.

In some embodiments, a GC content of a modified nucleic acid sequence ofthe present disclosure is increased relative to a reference and/or awild-type gene or coding sequence. The GC content of a modified nucleicacid is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%,at least 10%, at least 12%, at least 14%, at least 15%, at least 17%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70% greater than GC content of a wild type coding sequence. Insome embodiments, GC content is expressed as a percentage of G (guanine)and C (cytosine) nucleotides in the sequence.

In some embodiments, a codon adaptation index of a modified nucleic acidsequence of the present disclosure is at least 0.74, at least 0.76, atleast 0.77, at least 0.80, at least 0.85, at least 0.86, at least 0.87,at least 0.90, at least 0.95 or at least 0.98.

In some embodiments, a modified nucleic acid sequence of the presentdisclosure has a reduced level of CpG dinucleotides, that being areduction of about 10%, 20%, 30%, 50% or more, as compared to a wildtype or reference nucleic acid sequence. In some embodiments, a modifiednucleic acid has 1-5 fewer, 5-10 fewer, 10-15 fewer, 15-20 fewer, 20-25fewer, 25-30 fewer, 30-40 fewer, 40-45 fewer or 45-50 fewer, or evenfewer di-nucleotides than a reference sequence (e.g., a wild typesequence).

It is known that methylation of CpG dinucleotides plays an importantrole in the regulation of gene expression in eukaryotes. Specifically,methylation of CpG dinucleotides in eukaryotes essentially serves tosilence gene expression through interfering with the transcriptionalmachinery. As such, because of the gene silencing evoked by methylationof CpG motifs, nucleic acids and vectors having a reduced number of CpGdinucleotides will provide for high and longer-lasting transgeneexpression level.

Modified nucleic acid sequences may include flanking restriction sitesto facilitate subcloning into an expression vector. Many suchrestriction sites are well known in the art, and include, but are notlimited to Aval, Swal, ApaL1 and Xmal.

The present disclosure includes a modified nucleic acid of SEQ ID NO:1which encodes a functionally active fragment of the dystrophinpolypeptide. A “functionally active” or “functional dystrophinpolypeptide” indicates that the fragment provides the same or similarbiological function and/or activity as a full-length dystrophinpolypeptide. That is, the fragment provides the same function including,but not limited to, as a structural protein of myofilaments of a musclefiber. The biological activity of a functional fragment of dystrophinencompasses reversing or preventing the neuromuscular phenotypeassociated with Duchenne muscular dystrophy.

Thus, one embodiment of the invention relates to a method of purifying arAAV vector comprising a modified nucleic acid encoding amini-dystrophin protein, the nucleic acid comprising, consistingessentially of, or consisting of the nucleic acid sequence of SEQ IDNO:1 or a sequence at least about 90% identical thereto. In someembodiments, the nucleic acid is at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQID NO:1. In certain embodiments, the nucleic acid has a length that iswithin the capacity of a viral vector, e.g., a parvovirus vector, e.g.,a rAAV vector. In some embodiments, the nucleic acid is about 5000,4900, 4800, 4700, 4600, 4500, 4400, 4300, 4200, 4100, or about 4000nucleotides, or fewer.

In some embodiments, a nucleic acid encodes a mini-dystrophin proteincomprising, consisting essentially of, or consisting of the amino acidsequence of SEQ ID NO:2 or a sequence at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO:2.

In some embodiments, a nucleic acid encodes a deleted coppertransporting ATPase 2 protein comprising, consisting essentially of, orconsisting of the amino acid sequence of SEQ ID NO:15 or a sequence atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NO:15.

In some embodiments, a nucleic acid (e.g., SEQ ID NO:1) is part of arecombinant nucleic acid for production of dystrophin protein. Therecombinant nucleic acid may further comprise regulatory elements usefulfor increasing expression of dystrophin. In some embodiments, a nucleicacid is part of a recombinant nucleic acid for production of coopertransporting ATPase 2 protein. The recombinant nucleic acid may furthercomprise regulatory elements useful for increasing expression of coppertransporting ATPase 2.

Regulatory Elements

Methods of the present disclosure include purification of a rAAV vectorcomprising a recombinant nucleic acid including a modified nucleic acidencoding a polypeptide (e.g., mini-dystrophin) and various regulatory orcontrol elements. Typically, regulatory elements are nucleic acidsequence(s) that influence expression of an operably linkedpolynucleotide. The precise nature of regulatory elements useful forgene expression will vary from organism to organism and from cell typeto cell type including, for example, a promoter, enhancer, intron etc.,with the intent to facilitate proper heterologous polynucleotidetranscription and translation. Regulatory control can be affected at thelevel of transcription, translation, splicing, message stability, etc.Typically, a regulatory control element that modulates transcription isjuxtaposed near the 5′ end of the transcribed polynucleotide (i.e.,upstream). Regulatory control elements may also be located at the 3′ endof the transcribed sequence (i.e., downstream) or within the transcript(e.g., in an intron). Regulatory control elements can be located at adistance away from the transcribed sequence (e.g., 1 to 100, 100 to 500,500 to 1000, 1000 to 5000, 5000 to 10,000 or more nucleotides). However,due to the length of an AAV vector genome, regulatory control elementsare typically within 1 to 1000 nucleotides from the polynucleotide.

Promoter

As used herein, the term “promoter,” such as a “eukaryotic promoter,”refers to a nucleotide sequence that initiates transcription of aparticular gene, or one or more coding sequences (e.g., anmini-dystrophin coding sequence), in eukaryotic cells (e.g., a musclecell). A promoter can work with other regulatory elements or regions todirect the level of transcription of the gene or coding sequence(s).These regulatory elements include, for example, transcription bindingsites, repressor and activator protein binding sites, and othernucleotide sequences known to act directly or indirectly to regulate theamount of transcription from the promoter, including, for example,attenuators, enhances and silencers. The promoter is most often locatedon the same strand and near the transcription start site, 5′ of the geneor coding sequence to which it is operably linked. A promoter isgenerally 100-1000 nucleotides in length. A promoter typically increasesgene expression relative to expression of the same gene in the absenceof a promoter.

As used herein, a “core promoter” or “minimal promoter” refers to theminimal portion of a promoter sequence required to properly initiatetranscription. It may include any of the following: a transcriptionstart site, a binding site for RNA polymerase and a generaltranscription factor binding site. A promoter may also comprise aproximal promoter sequence (5′ of a core promoter) that contains otherprimary regulatory elements (e.g., enhancer, silencer, boundary element,insulator) as well as a distal promoter sequence (3′ of a corepromoter). In some embodiments, a core or minimal promoter is anα1-antitrypsin core or minimal promoter, optionally comprising orconsisting of the nucleic acid of SEQ ID NO:16.

Examples of a suitable promoter include adenoviral promoters, such asthe adenoviral major late promoter; heterologous promoters, such as thecytomegalovirus (CMV) promoter; the respiratory syncytial viruspromoter; the Rous Sarcoma Virus (RSV) promoter; the albumin promoter;inducible promoters, such as the Mouse Mammary Tumor Virus (MMTV)promoter; the metallothionein promoter; heat shock promoters; theα-1-antitrypsin promoter; the hepatitis B surface antigen promoter; thetransferrin promoter; the apolipoprotein A-1 promoter; chicken β-actin(CBA) promoter; the elongation factor 1a (EF1a) promoter; the hybridform of the CBA promoter (CBh promoter); the CAG promoter(cytomegalovirus early enhancer element and promoter, the first exon,and the first intron of chicken beta-actin gene and the splice acceptorof the rabbit beta-globin gene) (Alexopoulou et al. (2008) BioMed.Central Cell Biol. 9:2); a creatine kinase promoter; and the humandystrophin gene promoter.

In some embodiments, the promoter is a creatinine kinase promoter, e.g.,a promoter comprising, consisting essentially of, or consisting of thenucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4.

In some embodiments of the present disclosure, a eukaryotic promotersequence (e.g., a creatine kinase promoter) is operably linked to amodified nucleic acid encoding e.g., mini-dystrophin or a deleted coppertransporting ATPase2. In some embodiments, a promoter comprising thenucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:6 (e.g., a creatinekinase promoter) is operably linked to a modified nucleic acid encodingmini-dystrophin. In some embodiments, a promoter comprising a nucleicacid sequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99% or 100% identical to the nucleic acid sequenceof SEQ ID NO:3 or SEQ ID NO:4 is operably linked to a nucleic acidcomprising the nucleic acid sequence of SEQ ID NO:1. In someembodiments, a promoter comprising the nucleic acid sequence of SEQ IDNO:16 (e.g., an α1-antitrypsin promoter) is operably linked to amodified nucleic acid encoding a deleted copper transporting ATPase 2with deletion of MBS 1-4 (e.g., SEQ ID NO:15). In some embodiments, apromoter comprising a nucleic acid sequence at least 80%, at least 85%,at least 90%, at least 95%, at least 98%, at least 99% or 100% identicalto the nucleic acid sequence of SEQ ID NO:16 is operably linked to anucleic acid comprising the amino acid sequence of SEQ ID NO:15.

In some embodiments, a promoter comprising a nucleic acid sequence atleast 95% identical to the nucleic acid sequence of SEQ ID NO:3 or SEQID NO:4 is operably linked to a nucleic acid sequence at least 95%identical to the nucleic acid sequence of SEQ ID NO:1 and inducesexpression of a polypeptide encoded by the nucleic acid sequence of SEQID NO:1 in muscle cells.

A promoter may be constitutive, tissue-specific or regulated.Constitutive promoters are those which cause an operably linked gene tobe expressed essentially at all times. In some embodiments, aconstitutive promoter is active in most eukaryotic tissues under mostphysiological and developmental conditions.

Regulated promoters are those which can be activated or deactivated.Regulated promoters include inducible promoters, which are usually“off,” but which may be induced to turn “on,” and “repressible”promoters, which are usually “on,” but may be turned “off.” Manydifferent regulators are known, including temperature, hormones,cytokines, heavy metals and regulatory proteins. The distinctions arenot absolute; a constitutive promoter may often be regulated to somedegree. In some cases, an endogenous pathway may be utilized to provideregulation of the transgene expression, e.g., using a promoter that isnaturally downregulated when the pathological condition improves.

A tissue-specific promoter is a promoter that is active in only specifictypes of tissues, cells or organs. Typically, a tissue-specific promoteris recognized by transcriptional activator elements that are specific toa particular tissue, cell and/or organ. For example, a tissue-specificpromoter may be more active in one or several particular tissues (e.g.,two, three or four) than in other tissues. In some embodiments,expression of a gene modulated by a tissue-specific promoter is muchhigher in the tissue for which the promoter is specific than in othertissues. In some embodiments, there may be little, or substantially noactivity, of the promoter in any tissue other than the one for which itis specific. A promoter may be a tissue-specific promoter, such as themouse albumin promoter, or the transthyretin promoter (TTR), which areactive in liver cells. Other examples of tissue specific promotersinclude promoters from genes encoding skeletal α-actin, myosin lightchain 2A, dystrophin, muscle creatine kinase which induce expression inskeletal muscle (Li et al. (1999) Nat. Biotech. 17:241-245). Liverspecific expression may be induced using promoters from the albumin gene(Miyatake et al. (1997) J. Virol. 71:5124-5132), hepatitis B. virus corepromoter (Sandig, et al. (1996) Gene Ther. 3:1002-1009) andalpha-fetoprotein (Arbuthnot et al., (1996) Hum. Gene. Ther.7:1503-1514).

Enhancer

In another aspect, a modified nucleic acid encoding a therapeuticpolypeptide further comprises an enhancer to increase expression of thetherapeutic polypeptide. Typically, an enhancer element is locatedupstream of a promoter element but may also be located downstream orwithin another sequence (e.g., a transgene). An enhancer may be located100 nucleotides, 200 nucleotides, 300 nucleotides or more upstream ordownstream of a modified nucleic acid. An enhancer typically increasesexpression of a modified nucleic acid (e.g., encoding a therapeuticpolypeptide) beyond the increased expression provided by a promoterelement alone.

Many enhancers are known in the art, including, but not limited to, thecytomegalovirus major immediate-early enhancer. More specifically, thecytomegalovirus (CMV) MIE promoter comprises three regions: themodulator, the unique region and the enhancer (Isomura and Stinski(2003) J. Virol. 77(6):3602-3614). The CMV enhancer region can becombined with another promoter, or a portion thereof, to form a hybridpromoter to further increase expression of a nucleic acid operablylinked thereto. For example, a chicken β-actin (CBA) promoter, or aportion thereof, can be combined with a CMV promoter/enhancer, or aportion thereof, to make a version of CBA termed the “CBh” promoter,which stands for chicken beta-actin hybrid promoter, as described inGray et al. (2011, Human Gene Therapy 22:1143-1153). Like promoters,enhancers may be constitutive, tissue-specific or regulated.

In some embodiments of the present disclosure, a regulatory elementcomprises a hybrid enhancer and promoter, such as a synthetic hybridenhancer and promoter derived from the creatine kinase (CK) gene whichserves as a muscle specific transcription regulatory element (hCK) andwhich is operably linked to a modified nucleic acid encodingmini-dystrophin. In some embodiments, a synthetic hybrid enhancer andpromoter comprising the nucleic acid sequence of SEQ ID NO:5 is operablylinked to a modified nucleic acid encoding mini-dystrophin. In someembodiments, a synthetic hybrid enhancer and promoter derived from thecreatine kinase (CK) gene comprising a nucleic acid sequence at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99% or 100% identical to the nucleic acid sequence of SEQ ID NO:5 isoperably linked to a nucleic acid comprising the nucleic acid sequenceof SEQ ID NO:1.

In some embodiments, a synthetic hybrid enhancer and promoter derivedfrom the creatine kinase (CK) gene comprising a nucleic acid sequence atleast 95% identical to the nucleic acid sequence of SEQ ID NO:5 isoperably linked to a nucleic acid sequence at least 95% identical to thenucleic acid sequence of SEQ ID NO:1 and induces expression of apolypeptide encoded by the nucleic acid sequence of SEQ ID NO:1 inmuscle cells.

Fillers, Spacers and Stuffers

As disclosed herein, a recombinant nucleic acid intended for use in arAAV vector may include an additional nucleic acid element to adjust thelength of the nucleic acid to near, or at the normal size (e.g.,approximately 4.7 to 4.9 kilobases), of the viral genomic sequenceacceptable for AAV packaging into a rAAV vector (Grieger and Samulski(2005) J. Virol. 79(15):9933-9944). Such a sequence may be referred tointerchangeably as filler, spacer or stuffer. In some embodiments,filler DNA is an untranslated (non-protein coding) segment of nucleicacid. In some embodiments, a filler or stuffer polynucleotide sequenceis a sequence between about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60,60-70, 70-80, 80-90-90-100, 100-150, 150-200, 200-250, 250-300, 300-400,400-500, 500-750, 750-1000, 1000-1500, 1500-2000, 2000-3000 or more inlength.

AAV vectors typically accept inserts of DNA having a size ranging fromabout 4 kb to about 5.2 kb or about 4.1 to 4.9 kb for optimal packagingof the nucleic acid into the AAV capsid. In some embodiments, a rAAVvector comprises a vector genome having a total length between about 3.0kb to about 3.5 kb, about 3.5 kb to about 4.0 kb, about 4.0 kb to about4.5 kb, about 4.5 kb to about 5.0 kb or about 5.0 kb to about 5.2 kb. Insome embodiments, a rAAV vector comprises a vector genome having a totallength of about 4.5 kb. In some embodiments, a rAAV vector comprises avector genome that is self-complementary. While the total length of aself-complementary (sc) vector genome in a rAAV vector is equivalent toa single-stranded (ss) vector genome (i.e., from about 4 kb to about 5.2kb), the nucleic acid sequence (i.e., comprising the transgene,regulatory elements and ITRs) encoding the sc vector genome must be onlyhalf as long as a nucleic acid sequence encoding a ss vector genome inorder for the sc vector genome to be packaged in the capsid.

Introns and Exons

In some embodiments, a recombinant nucleic acid includes, for example,an intron, exon and/or a portion thereof. An intron may function as afiller or stuffer polynucleotide sequence to achieve an appropriatelength for vector genome packaging into a rAAV vector. An intron and/oran exon sequence can also enhance expression of a polypeptide (e.g., atransgene) as compared to expression in the absence of the intron and/orexon element (Kurachi et al. (1995) J. Biol. Chem. 270 (10):576-5281; WO2017/074526). Furthermore, filler/stuffer polynucleotide sequences (alsoreferred to as “insulators”) are well known in the art and include, butare not limited to, those described in WO 2014/144486 and WO2017/074526.

An intron element may be derived from the same gene as a heterologouspolynucleotide, or derived from a completely different gene or other DNAsequence (e.g., chicken β-actin gene, minute virus of mice (MVM)). Insome embodiments, a recombinant nucleic acid includes at least oneelement selected from an intron and an exon derived from a non-cognategene (i.e., not derived from the modified nucleic acid, e.g.,transgene).

Polyadenylation Signal Sequence (polyA)

Further regulatory elements may include a stop codon, a terminationsequence, and a polyadenylation (polyA) signal sequence, such as, butnot limited to a bovine growth hormone poly A signal sequence (BHGpolyA). A polyA signal sequence drives efficient addition of apoly-adenosine “tail” at the 3′ end of a eukaryotic mRNA which guidestermination of gene transcription (see, e.g., Goodwin and Rottman J.Biol. Chem. (1992) 267(23):16330-16334). A polyA signal acts as a signalfor the endonucleolytic cleavage of the newly formed precursor mRNA atits 3′ end and for addition to this 3′ end of an RNA stretch consistingonly of adenine bases. A polyA tail is important for the nuclear export,translation and stability of mRNA. In some embodiments, a poly A is aSV40 early polyadenylation signal, a SV40 late polyadenylation signal,an HSV thymidine kinase polyadenylation signal, a protamine genepolyadenylation signal, an adenovirus 5 E1b polyadenylation signal, agrowth hormone polyadenylation signal, a PBGD polyadenylation signal oran in silico designed polyadenylation signal.

In some embodiments, and optionally in combination with one or moreother regulatory elements described herein, a polyA signal sequence of arecombinant nucleic acid is a polyA signal that is capable of directingand effecting the endonucleolytic cleavage and polyadenylation of theprecursor mRNA resulting from the transcription of a modified nucleicacid encoding e.g., mini-dystrophin (e.g., SEQ ID NO:2) or a deletedcopper transporting ATPase 2 (e.g., SEQ ID NO:15). In some embodiments,a polyA sequence comprises or consists of the nucleic acid sequence ofSEQ ID NO:6 or SEQ ID NO:17. In some embodiments, a polyA sequencecomprises a nucleic acid sequence about 80%, about 85%, about 90%, about95%, about 98%, about 99% or 100% identical to the nucleic acid sequenceof SEQ ID NO:6 or SEQ ID NO:17. In some embodiments, a recombinantnucleic acid comprises at least one of: a promoter sequence (e.g., SEQID NO:3, SEQ ID NO:4), a hybrid enhancer and promoter (e.g., SEQ IDNO:5) and a polyA (SEQ ID NO:6) and modulates the expression of aheterologous polypeptide, optionally encoded by the nucleic acidsequence of SEQ ID NO:1. In some embodiments, a recombinant nucleic acidcomprises at least one of: a promoter sequence (e.g., SEQ ID NO:16), anda polyA (SEQ ID NO:17) and modulates the expression of a heterologouspolypeptide comprising the amino acid sequence of SEQ ID NO:15.

In some embodiments, a rAAV9 vector with tropism for muscle cells,contains a vector genome comprising AAV ITRs (e.g., AAV2 ITRs) and arecombinant nucleic acid comprising a modified (i.e., codon-optimized)nucleic acid encoding mini-dystrophin and at least one of the followingregulatory elements: a promoter (e.g., a human CK promoter), a hybridenhancer and a poly A signal sequence.

In some embodiments, a rAAV3B vector with tropism for liver cells,contains a vector genome comprising AAV ITRs (e.g., AAV2 ITRs) and arecombinant nucleic acid comprising a modified (i.e., codon-optimized)nucleic acid encoding a deleted copper transporting ATPase 2 (e.g.,amino acid sequence of SEQ ID NO:15) and at least one of the followingregulatory elements: a promoter (e.g., an α1-antitrypsin promoter, e.g.,of nucleic acid sequence SEQ ID NO:16) and a poly A signal sequence(e.g., nucleic acid of SEQ ID NO:17).

In some embodiments, a rAAV 9 vector with tropism for muscle cells,contains a vector genome comprising AAV ITRs (e.g., SEQ ID NO:7, SEQ IDNO:8) and a recombinant nucleic acid comprising a modified (i.e.,codon-optimized) nucleic acid (e.g., SEQ ID NO:1) encodingmini-dystrophin and at least one of the following regulatory elements: apromoter (e.g., SEQ ID NO:3 or SEQ ID NO:4), a hybrid enhancer andpromoter (e.g., SEQ ID NO:5) and a poly A (e.g., SEQ ID NO:6).

4. Assembly of Viral Vectors

A viral vector (e.g., rAAV vector) carrying a transgene (e.g., encodingmini-dystrophin) is assembled from a polynucleotide encoding atransgene, suitable regulatory elements and elements necessary forproduction of viral proteins which mediate cell transduction. Examplesof a viral vector include but are not limited to adenoviral, retroviral,lentiviral, herpesvirus and adeno-associated virus (AAV) vectors, and inparticular rAAV vector (as discussed, supra).

A vector genome component of a rAAV vector produced according to themethods of the disclosure include at least one transgene, e.g., a codonoptimized mini-dystrophin transgene and associated expression controlsequences for controlling expression of the modified nucleic acidencoding dystrophin, or a fragment thereof.

In an exemplary non-limiting embodiment, a vector genome includes aportion of a parvovirus genome, such as an AAV genome with rep and capdeleted and/or replaced by a modified nucleic acid (e.g., transgene,e.g., a codon optimized mini-dystrophin transgene) and its associatedexpression control sequences. A modified nucleic acid encodingdystrophin, or a fragment thereof, is typically inserted adjacent to oneor two (i.e., is flanked by) AAV ITRs or ITR elements adequate for viralreplication (Xiao et al. (1997) J. Virol. 71(2): 941-948), in place ofthe nucleic acid encoding viral rep and cap proteins. Other regulatorysequences suitable for use in facilitating tissue-specific expression ofa codon optimized mini-dystrophin transgene in the target cell (e.g.,muscle cell) may also be included.

Packaging Cell

One skilled in the art would appreciate that a rAAV vector comprising atransgene, and lacking virus proteins needed for viral replication(e.g., cap and rep), cannot replicate since such proteins are necessaryfor virus replication and packaging. Cap and rep genes may be suppliedto a cell (e.g., a host cell, e.g., a packaging cell) as part of aplasmid that is separate from a plasmid supplying the vector genome withthe transgene.

“Packaging cell” or “producer cell” means a cell or cell line which maybe transfected with a vector, plasmid or DNA construct, and provides intrans all the missing functions which are required for the completereplication and packaging of a viral vector. The required genes for rAAVvector assembly include the vector genome (e.g., a codon optimizedmini-dystrophin transgene, regulatory elements, and ITRs), AAV rep gene,AAV cap gene, and certain helper genes from other viruses such as, e.g.,adenovirus. One of ordinary skill would understand that the requisitegenes for AAV production can be introduced into a packaging cell invarious ways including, for example, transfection of one or moreplasmids. However, in some embodiments, some genes (e.g., rep, cap,helper) may already be present in a packaging cell, either integratedinto the genome or carried on an episome. In some embodiments, apackaging cell expresses, in a constitutive or inducible manner, one ormore missing viral functions.

Any suitable packaging cell known in the art may be employed in theproduction of a packaged viral vector. Mammalian cells or insect cellsare preferred. Examples of cells useful for the production of apackaging cell in the practice of the disclosure include, for example,human cell lines, such as PER.C6, WI38, MRC5, A549, HEK293 (whichexpress functional adenoviral E1 under the control of a constitutivepromoter), B-50 or any other HeLa cell, HepG2, Saos-2, HuH7, and HT1080cell lines. Suitable non-human mammalian cell lines include, forexample, VERO, COS-1, COS-7, MDCK, BHK21-F, HKCC or CHO cells.

In some embodiments, a packaging cell is capable of growing insuspension culture. In some embodiments, a packaging cell is capable ofgrowing in serum-free media. For example, HEK293 cells are grow insuspension in serum free medium. In another embodiment, a packaging cellis a HEK293 cell as described in U.S. Pat. No. 9,441,206 and depositedas American Type Culture Collection (ATCC) No. PTA 13274. Numerous rAAVpackaging cell lines are known in the art, including, but not limitedto, those disclosed in WO 2002/46359.

A cell line for use as a packaging cell includes insect cell lines. Anyinsect cell which allows for replication of AAV and which can bemaintained in culture can be used in accordance with the presentdisclosure. Examples include Spodoptera frugiperda, such as the Sf9 orSf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines,e.g., Aedes albopictus derived cell lines. A preferred cell line is theSpodoptera frugiperda Sf9 cell line. The following references areincorporated herein for their teachings concerning use of insect cellsfor expression of heterologous polypeptides, methods of introducingnucleic acids into such cells, and methods of maintaining such cells inculture: Methods in Molecular Biology, ed. Richard, Humana Press, NJ(1995); O'Reilly et al., Baculovirus Expression Vectors: A LaboratoryManual, Oxford Univ. Press (1994); Samulski et al. (1989) J. Virol.63:3822-3828; Kajigaya et al. (1991) Proc. Nat'l. Acad. Sci. USA 88:4646-4650; Ruffing et al. (1992) J. Virol. 66:6922-6930; Kimbauer et al.(1996) Virol. 219:37-44; Zhao et al. (2000) Virol. 272:382-393; and U.S.Pat. No. 6,204,059.

As a further alternative, viral vectors of the disclosure may beproduced in insect cells using baculovirus vectors to deliver therep/cap genes and rAAV template as described, for example, by Urabe etal. (2002) Human Gene Therapy 13:1935-1943. When using baculovirusproduction for AAV, in some embodiments, a vector genome isself-complementary. In some embodiments, a host cell is abaculovirus-infected cell (e.g., an insect cell) comprising, optionally,additional nucleic acids encoding baculovirus helper functions, therebyfacilitating production of a viral capsid.

A packaging cell generally includes one or more viral vector functionsalong with helper functions and packaging functions sufficient to resultin replication and packaging of the viral vector. These variousfunctions may be supplied together, or separately, to the packaging cellusing a genetic construct such as a plasmid or an amplicon, and they mayexist extrachromosomally within the cell line, or integrated into thehost cell's chromosomes. In some embodiments, a packaging cell istransfected with at least i) a plasmid comprising a vector genomecomprising a transgene and AAV ITRs and further comprising at least oneof the following regulatory elements: an enhancer, a promoter, an exon,an intron and a poly A and ii) a plasmid comprising a rep gene (e.g.,AAV2 rep) and a cap gene (e.g., AAV9 or other cap).

In some embodiments, a host cell is supplied with one or more of thepackaging or helper functions incorporated within, e.g., a host cellline with one or more vector functions incorporated extrachromosomallyor integrated into the cell's chromosomal DNA.

Helper Function

AAV is a dependovirus in that it cannot replicate in a cell withoutco-infection of the cell by a helper virus. Helper functions includehelper virus elements needed for establishing active infection of apackaging cell, which is required to initiate packaging of the viralvector. Helper viruses include, typically, adenovirus or herpes simplexvirus. Adenovirus helper functions typically include adenoviruscomponents adenovirus early region 1A (E1a), E1b, E2a, E4, and viralassociated (VA) RNA. Helper functions (e.g., E1a, E1b, E2a, E4, and VARNA) can be provided to a packaging cell by transfecting the cell withone or more nucleic acids encoding various helper elements.Alternatively, a host cell (e.g., a packaging cell) can comprise anucleic acid encoding the helper protein. For instance, HEK293 cellswere generated by transforming human cells with adenovirus 5 DNA and nowexpress a number of adenoviral genes, including, but not limited to E1and E3 (see, e.g., Graham et al. (1977) J. Gen. Virol. 36:59-72). Thus,those helper functions can be provided by the HEK 293 packaging cellwithout the need of supplying them to the cell by, e.g., a plasmidencoding them. In some embodiments, a packaging cell is transfected withat least i) a plasmid comprising a vector genome comprising a transgeneand AAV ITRs and further comprising at least one of the followingregulatory elements: an enhancer, a promoter, an exon, an intron and apoly A and ii) a plasmid comprising a rep gene (e.g., AAV2 rep) and acap gene (e.g., AAV9 or other cap) and iii) a plasmid comprising ahelper function.

Any method of introducing a nucleotide sequence carrying a helperfunction into a cellular host for replication and packaging may beemployed, including but not limited to, electroporation, calciumphosphate precipitation, microinjection, cationic or anionic liposomes,a carrier molecule (e.g., polyethylenimine (PEI)) and liposomes incombination with a nuclear localization signal. In some embodiments,helper functions are provided by transfection using a virus vector, orby infection using a helper virus, standard methods for producing viralinfection may be used.

The vector genome may be any suitable recombinant nucleic acid, such asa DNA or RNA construct and may be single stranded, double stranded, orduplexed (i.e., self-complementary as described in WO 2001/92551).

4. Production of Packaged Viral Vector

Viral vectors can be made by several methods known to skilled artisans(see, e.g., WO 2013/063379). An exemplary non-limiting method isdescribed in Grieger, et al. (2015) Molecular Therapy 24(2):287-297, thecontents of which are incorporated by reference herein for all purposes.Briefly, efficient transfection of HEK293 cells is used as a startingpoint, wherein an adherent HEK293 cell line from a qualified clinicalmaster cell bank is used to grow in animal component-free suspensionconditions in shaker flasks and WAVE bioreactors that allow for rapidand scalable rAAV production. Using a triple transfection method (e.g.,WO 96/40240), a HEK293 cell line suspension can generate greater than1×10⁵ vector genome containing particles (VG)/cell, or greater than1×10¹⁴ VG/L of cell culture, when harvested 48 hours post-transfection.More specifically, triple transfection refers a method whereby apackaging cell is transfected with three plasmids: one plasmid encodesthe AAV rep and cap (e.g., AAV9 cap) genes, another plasmid encodesvarious helper functions (e.g., adenovirus or HSV proteins such as E1a,E1b, E2a, E4, and VA RNA, and another plasmid encodes a transgene (e.g.,dystrophin, or a fragment thereof) and various elements to controlexpression of the transgene.

Single-stranded vector genomes are packaged into capsids as the plusstrand or minus strand in about equal proportions. In some embodimentsof a rAAV vector, a vector genome is in the plus strand polarity (i.e.,the sense or coding sequence of the DNA strand). In some embodiments arAAV vector, a vector is in the minus strand polarity (i.e., theantisense or template DNA strand). Given the nucleotide sequence of aplus strand in its 5′ to 3′ orientation, the nucleotide sequence of aminus strand in its 5′to 3′ orientation can be determined as thereverse-complement of the nucleotide sequence of the plus strand.

To achieve the desired yields, a number of variables are optimized suchas selection of a compatible serum-free suspension media that supportsboth growth and transfection, selection of a transfection reagent,transfection conditions and cell density.

A rAAV vector may be purified by methods standard in the art such as bycolumn chromatography or cesium chloride gradients. Methods forpurifying rAAV vectors are known in the art and include methodsdescribed in Clark et al. (1999) Human Gene Therapy 10(6):1031-1039;Schenpp and Clark (2002) Methods Mol. Med. 69:427-443; U.S. Pat. No.6,566,118 and WO 98/09657.

After rAAV vectors of the present disclosure have been produced andpurified according to methods disclosed herein, they can be titered(e.g., the amount of rAAV vector in a sample can be quantified) toprepare compositions for administration to subjects, such as humansubjects with Duchenne muscular dystrophy. rAAV vector titering can beaccomplished using methods know in the art.

In some embodiments, the number of viral particles, including particlescontaining a vector genome and “empty” capsids that do not contain avector genome, can be determined by electron microscopy, e.g.,transmission electron microscopy (TEM). Such a TEM-based method canprovide the number of vector particles (or virus particles in the caseof wild type AAV) in a sample. In some embodiments, the amount ofparticles, containing a vector genome (full capsids), and “empty”capsids that do not contain a vector genome, can be determined by chargedetection mass spectrometry, analytical ultracentrifugation (AUC),and/or measurement of absorbance at 260 nm and 280 nm to determineA260/A280 ratio.

In some embodiments, rAAV vector genomes can be titered usingquantitative PCR (qPCR) using primers against any sequence in the vectorgenome, for example ITR sequences (e.g., SEQ ID NO:7 or SEQ ID NO:8),and/or sequences in the transgene (or regulatory elements). Byperforming qPCR in parallel on dilutions of a standard of knownconcentration, such as a plasmid containing the sequence of the vectorgenome, a standard curve can be generated permitting the concentrationof the rAAV vector to be calculated as the number of vector genomes (VG)per unit volume such as microliters or milliliters. By comparing thenumber of vector particles as measured by, e.g., SEC or ELISA, to thenumber of vector genomes in a sample, the percentage of empty capsidscan be estimated. Because the vector genome contains the therapeutictransgene, vg/kg or vg/ml of a vector sample may be more indicative ofthe therapeutic amount of the vector that a subject will receive thanthe number of vector particles, some of which may be empty and notcontain a vector genome. Once the concentration of rAAV vector genomesin the stock solution is determined, it can be diluted into or dialyzedagainst suitable buffers for use in preparing a composition (e.g., adrug substance) for administration to subjects (e.g., subjects withDuchenne muscular dystrophy).

5. rAAV Vector Purification by Anion-Exchange Chromatography (AEX)

A novel, universal purification strategy, based on ion exchangechromatography methods, may be used to generate high purity rAAV vectorpreparations of various AAV serotypes and/or from chimeric capsids(e.g., AAV1, AAV2, AAV3 including AAV3A and AAV3B, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV12, AAVrh10, AAVrh74, avian AAV, bovine AAV,canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, andrecombinantly produced variants (e.g., capsid variants with insertions,deletions and substitutions, etc.), such as variants referred to asAAV2i8, NP4, NP22, NP66, AAVDJ, AAVDJ/8, AAVDJ/9, AAVLK03, RHM4-1,AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8,AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15).In some embodiments, this process can be completed in less than a week,result in high full to empty capsid ratios (up to 70% full capsids),provide step yields up to 70% and purity suitable for clinical use. Insome embodiments, such a method is universal with respect to AAVserotype and/or chimerism of the capsid. Scalable manufacturingtechnology, as described herein, may be used to manufacture GMP clinicaland commercial grade rAAV vectors to treat disease (e.g., DMD,Friedreich's Ataxia, Wilson Disease etc.).

Production of recombinant AAV vector (rAAV) for gene therapy requirespurification of the rAAV vector from a host cell (e.g., host cell debrisincluding but not limited to host cell DNA, RNA, proteins, lipids,membrane and organelles) that produced the vector, as well as removal ofcapsids that do not contain a complete vector genome (e.g., intermediateand/or empty capsids) and thus, do not comprise a therapeutic transgene.

Such purification methods generally comprise multiple steps including,for example, lysis of the host cell, precipitation of cellular proteinand DNA, separation of the rAAV vector from host cell protein andnucleic acids, and separation of the rAAV vector from empty andintermediate capsids by column purification, low speed centrifugation,ultracentrifugation, normal flow filtration,ultrafiltration/diafiltration or any combination of these methods.Column purification may include, for example, ion exchangechromatography (e.g., anion, cation), affinity chromatography, sizeexclusion chromatography, multimodal chromatography, and/or hydrophobicinteraction chromatography. Centrifugation methods may include, forexample, ultracentrifugation or low speed centrifugation (e.g., forremoval of solids and clarification). Filtration methods may include,for example, diafiltration, depth filtration, nominal filtration and/orabsolute filtration.

AEX employs a positively charged stationary phase (e.g., a resin) toseparate substances (e.g., AAV capsids, DNA, protein, high molar massspecies, amino acids) based on charge differences of said substances,and is useful for separating rAAV capsids from impurities based oncharge differences at moderately acidic to alkaline pH (e.g., greaterthan pH 6). AEX can also separate empty capsids from rAAV vectorscontaining a complete vector genome (i.e., full capsid) by relying onthe charge differences of empty capsids as compared to full capsids.

Without wishing to be bound by theory, the tightness of binding betweenan AAV capsid and an AEX chromatography stationary phase is related tothe strength of the negative charge of the capsid, including the chargecontribution from any nucleic acid within the capsid, solution pH andsolution conductivity (Qu, G. et al., J. Virological. Methods (2007)140:183-192). In some embodiments, an AEX chromatography stationaryphase is a resin comprising polystyrenedivinylbenzene particles modifiedwith covalently bound quaternized polyethyleneimine, and optionally OHgroups (e.g., POROS™ 50 HQ resin). Polystyrenedivinylbenzene particlesmay comprise pores of 500-10,000 Angstroms (Å).

In some embodiments, an AEX chromatography stationary phase is a resincomprising agarose particles with a cationic ligand (e.g. Capto QImpRes, Q Sepharose High Performance). In some embodiments, an AEXchromatography stationary phase is a resin selected from the groupconsisting of Capto Q, Capto Q XP, Q Sepharose XL, STREAMLINE Q XL,Capto HiRes Q, RESOURCE Q, SOURCE 15 Q, SOURCE 30 Q, Q Sepharose HP, QSepharose FF, Q Sepharose™ BB, POROS™ 20 HQ, POROS™ XQ, TOYOPEARLQAE-550C, TOYOPEARL Q-600C AR, TOYOPEARL GigaCap Q-650S, TOYOPEARLGigaCap Q-650M, TOYOPEARL SuperQ-650S, TOYOPEARL SuperQ-650M, TOYOPEARLSuperQ-650C, TSKgel SuperQ-5PW (20), TSKgel SuperQ-5PW (30), Q CeramicHyperD F, ESHMUNO® Q, Fractogel® EMD TMAE (S), Fractogel® EMD TMAE (M),Fractogel® EMD TMAE Hicap (M), Fractogel® EMD TMAE (S), Fractogel® EMDTMAE (M), Fractogel® EMD TMAE Hicap (M), Nuvia Q, Nuvia HP-Q, UNOsphereQ, Macro-Prep High Q, Macro-Prep 25 Q, BioRad AG® 1-X2, WorkBeads™ 40Q,WorkBeads™ 100Q, Cellufine MAX Q-r, Cellufine MAX Q-h, Praesto™ Q65,Praesto™ Q90, Praesto™ Jetted Q35, BAKERBOND™ POLYQUAT, BAKERBOND™POLYPEI, YMC—BioPro Q30, YMC—BioPro Q75, YMC—BioPro SmartSep 010,YMC—BioPro SmartSep Q30, DEAE Sepharose FF, ANX Sepharose 4 FF (highsub), POROS™ 50 PI, POROS™ 50 D, TOYOPEARL NH₂-750F, TOYOPEARL GigaCapDEAE-650M, TOYOPEARL DEAE-650S, TOYOPEARL DEAE-650M, TOYOPEARLDEAE-650C, TSKgel DEAE-5PW (20), TSKgel DEAE-5PW (30), Ceramic HyperDDEAE, Hypercel Star AX, Fractogel® EMD DEAE (M), Fractogel® EMD DMAE (M)Resin, Macro-Prep DEAE, WorkBeads™ 40 DEAE, Cellufine MAX DEAE, DEAEPuraBead HF and WorkBeads™ 40 TREN.

In some embodiments, an AEX chromatography stationary phase is amonolith comprising porous poly-methacrylate with a cationic ligand(e.g. ClMmultus™ QA). In some embodiments, an AEX chromatographystationary phase is a membrane adsorber comprising polyethersulfone witha cationic ligand (e.g. Mustang Q, Mustang E, Sartobind® Q, SartobindSTIC® PA).

In some embodiments, a rAAV vector can be purified by AEX from asolution exiting from an affinity chromatography stationary phase (e.g.,“eluting from the stationary phase”) comprised of mobile phase andmaterial such as rAAV vector or capsid that passed through thestationary phase or was displaced from the stationary phase. Thissolution may be referred to as an affinity eluate or an “affinity pool.”

In some embodiments, a rAAV vector can be purified by AEX from a“supernatant from a cell lysate” (also known as a “clarified lysate”),which, as used herein, refers to a solution collected followingsedimentation of lysed host cells from a host cell culture.

In some embodiments, a rAAV vector can be purified by AEX from a“post-harvest solution”, which, as used herein, refers to solutionresulting from a cell lysis that has undergone flocculation, depthfiltration and/or nominal filtration.

In some embodiments, a rAAV vector can be purified from a solutionhaving undergone at least one other purification or processing step(e.g., cell lysis, flocculation, filtration, dilution, pH adjustment,chromatography). In some embodiments, an affinity eluate has beendiluted, and optionally filtered prior to purification of the rAAVvector, such as prior to loading the affinity eluate onto the AEXcolumn.

In some embodiments, a rAAV vector can be purified by AEX from anaffinity eluate, optionally having undergone at least one otherpurification or processing step (e.g., cell lysis, flocculation,filtration, dilution, pH adjustment, chromatography). In someembodiments, a rAAV vector can be purified by AEX from a cell lysate,optionally having undergone at least one other purification orprocessing step (e.g., cell lysis, flocculation, filtration, dilution,pH adjustment, chromatography). In some embodiments, a rAAV vector canbe purified by AEX from a post-harvest solution, optionally havingundergone at least one other purification or processing step (e.g., celllysis, flocculation, filtration, dilution, pH adjustment,chromatography).

As a solution comprising a substance to be purified (e.g., a rAAVvector) and impurities, flows through an AEX stationary phase, asubstance that binds (e.g., negatively charged proteins such as an AAVcapsid or rAAV vector) to a positively charged AEX stationary phase areretained within the stationary phase. Unbound substances pass throughthe column and are collected in a flow-through, and/or during asubsequent wash step. Bound substances may be eluted from the stationaryphase by adjusting a salt concentration and/or pH within the column. Forexample, and without wishing to be bound by any particular theory ofoperation, a salt concentration of an elution buffer is graduallyincreased such that anions in the salt (e.g., acetate (C₂H₃O₂ ⁻), Cl⁻SO₄⁻²) compete with and displace (i.e., elute) a substance bound to theresin. In another embodiment, the pH of the solution within the columncan be gradually decreased to decrease the negative charge of a boundsubstance and cause it to be released (i.e., eluted) from the stationaryphase. Upon release from the stationary phase, a substance may becollected as a column eluate.

Without wishing to be bound by theory, separation of substances, such asa mixture of AAV capsids, or more specifically a mixture of a rAAVvector (i.e., a full capsid), an AAV capsid (e.g., an empty capsid, anintermediate capsid) and host cell proteins, will depend on the totalcharge difference of the substances. The charge composition of ionizableside groups will determine the total charge of a protein at a particularpH. At the isoelectric point (pl), the total charge on a protein is 0and it will not bind to a matrix. If the pH is above the pl, a proteinwill have a negative charge and bind to an anion exchange columnstationary phase.

An AEX protocol for separation of full rAAV vectors from empty capsidsincludes multiple steps, for example, pre-use flushing of a column mediato displace storage solution, pre-use sanitizing of a column stationaryphase, post-use sanitizing of a column stationary phase, equilibrating acolumn stationary phase, loading a solution (e.g., a diluted affinityeluate) comprising a rAAV vector onto a column stationary phase, elutinga substance to be purified from a stationary phase (e.g., by gradientelution, by step elution), applying a gradient hold to a columnstationary phase, sanitizing a column stationary phase, regenerating acolumn stationary phase, applying a storage solution to a columnstationary phase. One of skill in the art will understand that an AEXprotocol for purification of rAAV vectors may comprise all, or only someof these steps. One of skill in the art will also understand that theorder of these steps may vary, and that certain steps may be performedmore than once, and not necessarily in sequence.

AEX Column Preparation

AEX methods of the disclosure may be performed at various scalesutilizing columns ranging in volume from 1.0 mL to 20 L. In someembodiments, an AEX method includes use of a column with a column volume(CV) of about 1.0 mL, about 5.1 mL, about 49 mL, about 52 mL, about 6.67mL, about 1.256 L, about 1.3 L, about 6.0 L, about 6.1 L, about 6.2 L,about 6.3 L, about 6.4 L, about 6.5 L, about 6.6 L, about 6.7 L, about6.8 L, about 6.9 L, or about 7.0 L. In some embodiments, an AEX methodof the disclosure includes use of a column with a CV of 1.0 mL to 20 L,e.g., 1.0 ml to 10 mL, 30 mL to 70 mL, 10 mL to 100 mL, 100 mL to 1000mL, 1 L to 1.5 L, 1.5 L to 2.0 L, 2.0 L to 5 L, 5 L to 7.5 L, 7.5 L to10 L, 10 L to 15 L or 15 L to 20 L. In some embodiments, an AEX methodof the disclosure includes use of a column with a CV of 1.0 mL to 10 L,10 mL to 10 L, 100 mL to 20 L, 100 mL to 10 L, 1 L to 20 L, 1L to 10 L,1 L to 5 L, 1 L to 2 L or 1 L to 1.5 L. In some embodiments, an AEXmethod of the disclosure includes use of a column with a CV of 6.0 L to6.6 L (e.g., 6.4 L).

A volume of solution applied to a column to, for example, to equilibratea stationary phase therein, is generally expressed in terms of a “columnvolume” (CV), with one CV equivalent to the volume of the column.

In some embodiments, an AEX chromatography stationary phase (alsoreferred to herein as “resin” or “media”) of the disclosure is apolystyrenedivinylbenzene particle with covalently bound quaternizedpolyethyleneimine (e.g., POROS™ 50 HQ resin).

Generally, prior to application (i.e., loading) of a solution to bepurified (e.g., an affinity chromatography eluate, also referred toherein as an “affinity eluate” or an “affinity pool”) to a columncomprising a chromatography stationary phase, at least one solution isapplied to the stationary phase to, for example, flush, sanitize,regenerate and/or equilibrate the stationary phase. In some embodiments,an “affinity eluate” or an “affinity pool” has been diluted, andoptionally filtered prior to loading of the solution onto the AEXcolumn.

As disclosed herein, a method of preparing an AEX stationary phase foruse in a method of purifying a rAAV (e.g., rAAV9, rAAV3B or others)vector from a solution (e.g., an affinity eluate) by AEX comprisespre-use flushing of the AEX stationary phase in a column. In someembodiments, pre-use flushing of the AEX stationary phase is intended todisplace a storage solution (e.g., a solution comprising ethanol) fromthe stationary phase. In some embodiments, pre-use flushing of a columnprecedes loading a solution comprising a rAAV vector to be purified ontothe column. In some embodiments, pre-use flushing comprises applicationof water (e.g., water for injection) to AEX stationary phase in acolumn. In some embodiments, pre-use flushing comprises an upward flowof water. During upward flow of pre-use flushing, the flow direction isopposite that of chromatographic separation steps (e.g., loading,washing or eluting), such that the solution (e.g., water) flows from thebottom of the column to the top of the column, whereas during achromatographic separation step (e.g., loading) the solution flows fromthe top of the column to the bottom of the column. In some embodiments,pre-use flushing comprises application of 1 to 10 column volumes (CV)(e.g., about 5 CV) of water to AEX stationary phase in a column, at alinear velocity of 10 cm/hr to 1000 cm/hr and/or a flow rate of 0.2L/min to 3.0 L/min. In some embodiments, pre-use flushing comprisesapplication of ≥4.5 CV (e.g., about 5 CV) of water for injection to AEXstationary phase in a column, at a linear velocity of 270 cm/hr to 330cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5 L/min to 2.0 L/min(e.g., about 1.8 L/min) and/or a residence time (i.e., a contact time)of 3.5 min/CV to 4.5 min/CV (e.g., about 4 min/CV).

A method of preparing an AEX stationary phase for use in a method ofpurifying a rAAV (e.g., rAAV9, rAAV3B or others) vector from a solution(e.g., an affinity eluate) by AEX comprises sanitizing the AEXstationary phase in a column. Sanitizing an AEX stationary phase servesto reduce the bioburden (including, but not limited to bacteria) and/orinactivate microbes and viruses within the column, and more generally toremove contaminants such as proteins, particulates, etc. In someembodiments, sanitizing precedes loading a solution comprising a rAAVvector to be purified onto a column. In some embodiments, sanitizingcomprises application of a solution comprising NaOH, ethanol, aceticacid, phosphoric acid, guanidine HCl, urea, PAB (phosphoric add, aceticacid, benzyl alcohol), peracetic acid etc. to an AEX stationary phase ina column. In some embodiments, sanitizing comprises application of asolution comprising 0.1 M to 1.0 M, about 0.1 M to about 0.8 M, about0.1 M to about 0.6 M, about 0.2 M to about 0.8 M, about 0.2 M to about0.6 M or about 0.4 M to about 0.6 M (e.g., about 0.5 M) NaOH to AEXstationary phase in a column. In some embodiments, sanitizing comprisesapplication a solution comprising about 0.5 M NaOH to AEX stationaryphase in a column using an upward flow (i.e., that is the flow directionis opposite that of chromatographic separation steps, e.g., loading,washing or eluting). In some embodiments, sanitizing comprisesapplication a solution comprising about 0.5 M NaOH to AEX stationaryphase in a column using an downward flow (i.e., that is the flowdirection is in the same direction as that of chromatographic separationsteps, e.g., loading, washing or eluting). In some embodiments,sanitizing comprises application of 14.4 CV to 17.6 CV (e.g., about 16CV) of a solution comprising about 0.5 M NaOH to AEX stationary phase ina column. In some embodiments, sanitizing comprises application of 5 CVto 10 CV (e.g., about 8 CV) of a solution comprising about 0.5 M NaOH toAEX stationary phase in a column. In some embodiments, sanitizingcomprises application of 5 CV to 20 CV of a solution comprising about0.5 M NaOH to an AEX stationary phase in a column at a linear velocityof 100 cm/hr to 1000 cm/hr and/or a flow rate of 0.2 L/min to 3.0 L/min.In some embodiments, sanitizing comprises application of 14.4 CV to 17.6CV (e.g. about 16 CV) of a solution comprising about 0.5 M NaOH to AEXstationary phase in a column at a linear velocity of 270 cm/hr to 330cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5 L/min to 2.0 L/min(e.g., about 1.8 L/min) and/or a residence time (i.e., the amount oftime per column volume that the solution is in contact with thestationary phase within the column, and also referred to herein as thecontact time) of 3.5 min/CV to 4.5 min/CV (e.g., about 4 min/CV). Insome embodiments, sanitizing comprises application of 5 CV to 10 CV(e.g. about 8 CV) of a solution comprising about 0.5 M NaOH to AEXstationary phase in a column at a linear velocity of 270 cm/hr to 330cm/hr (e.g., about 298 cm/hr) and/or a residence time of 1.5 min/CV to2.5 min/CV (e.g., about 2 min/CV).

A method of preparing an AEX stationary phase for use in a method ofpurifying a rAAV (e.g., rAAV9, rAAV3B or others) vector from a solution(e.g., an affinity eluate) by AEX comprises regenerating (also referredto herein as “a rinse”) an AEX stationary phase in a column. One ofskill in the art will understand that regenerating an ion exchangestationary phase serves to replace ions taken up in the exchange processwith the original ions that occupied the exchange sites. In someembodiments, regeneration can also refer to bringing back a stationaryphase to its original state by, for example, the removal of impuritiesusing a strong solvent. In some embodiments, regenerating precedesloading a solution comprising a rAAV vector to be purified onto astationary phase. In some embodiments, regenerating may be performed ona stationary phase more than once.

In some embodiments, regenerating comprises application of a solutioncomprising a salt and/or a buffering agent, with a pH ranging from 8 to10, to an AEX stationary phase in a column. In some embodiments, a saltis selected from the group consisting of sodium chloride (NaCl), sodiumacetate (NaAcetate,CH₃COONa), ammonium acetate (NH₄Acetate), magnesiumchloride (MgCl₂) or sodium sulfate (Na₂SO₄). In some embodiments, aconcentration of a salt in a solution (e.g., NaCl) ranges from 1 M to 5M (e.g., about 1 M to about 4.5 M, about 1 to about 4M, about 1 M toabout 3.5 M, about 1M to about 3 M, about 1M to about 2.5 M or about 1.5M to about 2.5 M. In some embodiments, a concentration of a salt in asolution (e.g., NaCl) is about 1 M, about 2 M, about 3 M, about 4 M orabout 5 M. In some embodiments, regenerating comprises application of asolution comprising 1 M to 3 M (e.g., 2 M) NaCl to the stationary phasein the column.

In some embodiments, a buffering agent is selected from the groupconsisting of Tris (e.g., a mixture of Tris Base and Tris-HCl), BIS-Trispropane, and/or bicine. In some embodiments, the concentration of thebuffering agent (e.g., Tris) in a solution ranges from 10 mM to 500 mM(e.g., about 10 mM to about 450 mM, about 10 mM to about 400 mM, about10 mM to about 350 mM, about 10 mM to about 300 mM, about 10 mM to about250 mM, about 10 mM to about 200 mM, about 10 mM to about 150 mM, orabout 50 mM to about 150 mM. In some embodiments, the concentration ofthe buffering agent (e.g., Tris) in a solution is about 10 mM, about 20mM about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 300 mM,about 400 mM or about 500 mM. In some embodiments, regeneratingcomprises application of a solution comprising 50 mM to 150 mM (e.g.,100 mM) Tris to a stationary phase in a column.

In some embodiments, regenerating comprises application of a solutionwith a pH of about 7 to 11 (e.g., about 7.5 to 10.5, about 8 to 10, orabout 7, 7.5, 8, 8,5, 9, 9.5, 10, 10.5 or 11) to a stationary phase in acolumn.

In some embodiments, regenerating comprises application of a solutioncomprising about 1 M to 3 M (e.g., about 2 M) NaCl, 50 mM to 150 mM(e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to AEXstationary phase in a column. In some embodiments, regeneratingcomprises application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solutioncomprising about 2 M NaCl, 100 mM Tris, pH 9 to AEX stationary phase ina column. In some embodiments, regenerating comprises application of 1to 10 CV of a solution comprising about 2 M NaCl, 100 mM Tris, pH 9 toAEX stationary phase in a column at a linear velocity of 100 to 1000cm/hr and/or a flow rate of 0.2 to 3.0 L/min. In some embodiments,regenerating comprises application of 4.5 to 5.5 (e.g., about 5) CV of asolution comprising about 2 M NaCl, 100 mM Tris, pH 9 to AEX stationaryphase in a column at a linear velocity of 270 to 330 cm/hr (e.g., about298 cm/hr, about 300 cm/hr), a flow rate of 1.5 to 2.0 L/min (e.g.,about 1.8 L/min) and/or a residence time (i.e., a contact time) of 1.5to 4.5 min/CV (e.g., about 2 min/CV, about 4 min/CV).

In some embodiments, the present disclosure provides a method ofpreparing an AEX stationary phase for use in a method of purifying arAAV (e.g., rAAV9, rAAV3B or others) vector by AEX, the methodcomprising a step of: i) pre-use flushing comprising application of ≥4.5CV (e.g., about 5 CV) of water for injection to AEX stationary phase ina column; ii) sanitizing comprising application of about 5 CV to 10 CV(e.g., about 8 CV) or about 14.4 to 17.6 CV (e.g., about 16 CV) of asolution comprising 0.1 M to 1.0 M (e.g., about 0.5 M NaOH) to the AEXstationary phase in the column, optionally by upward flow; and/or iii)regenerating comprising application of 4.5 to 5.5 CV (e.g., about 5 CV)of a solution comprising 1 M to 3 M (e.g., about 2 M) NaCl, 50 mM to 150mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to the AEXstationary phase in the column; wherein at least one of steps i)-iii) isperformed at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about298 cm/hr, about 300 cm/hr), a flow rate of 1.5 L/min to 2.0 L/min(e.g., about 1.8 L/min) and/or a residence time of 1.5 min/CV to 4.5min/CV (e.g., about 2 min/CV, about 4 min/CV); optionally wherein atleast one step is performed prior to loading a solution comprising therAAV vector to be purified onto the column; and optionally wherein theAEX stationary phase is POROS™ 50 HQ. One of ordinary skill willunderstand that the above steps may be performed in any order and may beperformed more than once.

Equilibration

A method of preparing an AEX stationary phase for use in a method ofpurifying a rAAV (e.g., rAAV9, rAAV3B or others) vector from a solution(e.g., an affinity eluate) by AEX comprises equilibration of the AEXstationary phase in a column. In some embodiments, equilibration of anAEX stationary phase in a column serves to adjust the pH, conductivity,modifier (e.g., salt, detergent, amino acid etc.) concentration, orother condition, of the mobile and stationary phase such that somesubstances loaded onto the column will bind to the stationary phase, andothers will flow through with the mobile phase. For example, conditionswithin the column may be adjusted by the application of a series ofequilibration buffers to the column such that full rAAV vectors bind tothe stationary phase, and at least a portion of the empty capsids do notbind. In some embodiments, AEX stationary phase in a column isequilibrated prior to application of a solution comprising a substanceto be purified (e.g., a rAAV vector) to the column. In some embodiments,AEX stationary phase in a column is equilibrated by application of anequilibration buffer (e.g., a first equilibration buffer, a secondequilibration buffer, a third equilibration buffer, a fourthequilibration buffer, etc.). An equilibration buffer may also bereferred to herein as a “wash buffer,” a “post-sanitization rinse,” a“rinse,” or a “regeneration buffer.” Reference to an equilibrationbuffer as a first, second, third, fourth, etc. equilibration buffer doesnot necessarily imply the order in which the buffers are applied to acolumn.

In some embodiments, an equilibration buffer (e.g., a firstequilibration buffer, a second equilibration buffer, a thirdequilibration buffer, a fourth equilibration buffer, etc.) comprises atleast one component selected from the group consisting of a bufferingagent, a salt, an amino acid, a detergent and/or a combination thereof.In some embodiments, a buffering agent is Tris (e.g., a mixture of TrisBase and Tris-HCl), BIS-Tris propane, diethanolamine, diethylamine,tricine, triethanolamine and/or bicine. One of ordinary skill in the artwould understand that a Tris buffer with a desired pH can be preparedusing Tris Base, Tris-HCl or both. In some embodiments, a salt is sodiumchloride (NaCl), sodium acetate (NaAcetate (CH₃COONa)), ammonium acetate(NH₄Acetate), magnesium chloride (MgCl₂) or sodium sulfate (Na₂SO₄). Insome embodiments, a salt is sodium acetate. In some embodiments, anamino acid is histidine, arginine, glycine or citrulline. In someembodiments, a detergent is poloxamer 188 (P188), Triton X-100,Polysorbate 80, Brij-35 or nonyl phenoxypolyethoxylethanol (NP-40).

In some embodiments, an equilibration buffer comprises 10 mM to 350 mMof a buffering agent selected from the group consisting of Tris (e.g., amixture of Tris Base and Tris-HCl), BIS-Tris propane, diethanolamine,diethylamine, tricine, triethanolamine and bicine. In some embodiments,an equilibration buffer comprises 10 mM to 350 mM, 10 mM to 300 mM Tris,10 mM to 250 mM Tris, 10 mM to 200 mM Tris, 10 mM to 150 mM Tris, 10 mMto 100 mM Tris or 10 mM to 50 mM Tris. In some embodiments, anequilibration buffer comprises 30 mM to 350 mM Tris, 30 mM to 300 mMTris, 30 mM to 250 mM Tris, 30 mM to 2000 mM Tris, 30 mM to 150 mM Tris,30 mM to 100 mM Tris. In some embodiments, an equilibration buffercomprises 50 mM to 300 mM Tris, 50 mM to 250 mM Tris, 50 mM to 200 mMTris, 50 mM to 150 mM Tris. In some embodiments, an equilibration buffercomprises 100 mM to 350 mM Tris, 100 mM to 250 mM Tris or 100 mM to 150mM Tris. In some embodiments, an equilibration buffer comprises about 10mM Tris, about 20 mM Tris, about 30 mM Tris, about 40 mM Tris, about 50mM Tris, about 60 mM Tris, about 70 mM Tris, about 80 mM Tris, about 90mM Tris, about 100 mM Tris, about 110 mM Tris, about 120 mM Tris, about130 mM Tris, about 140 mM Tris, about 150 mM Tris, about 160 mM Tris,about 170 mM Tris, about 180 mM Tris, about 190 mM Tris, about 200 mMTris, about 220 mM Tris, about 240 mM Tris, about 250 mM Tris, about 275mM Tris, about 300 mM Tris or about 350 mM Tris. In some embodiments, anequilibration buffer comprises about 20 mM Tris, 100 mM Tris or 200 mMTris.

In some embodiments, an equilibration buffer comprises 1 mM to 1M salt,and preferably about 500 mM salt. In some embodiments, an equilibrationbuffer comprises about 10 mM to about 950 mM, about 10 mM to about 900mM, about 10 mM to about 850 mM, about 10M to about 800 mM, 10 mM toabout 750 mM, about 10 mM to about 700 mM, about 10 mM to about 650 mM,about 10 mM to about 600 mM, about 10 mM to about 550 mM, about 50 mM toabout 750 mM, about 50 mM to about 700 mM, about 50 mM to about 650 mM,about 50 mM to about 600 mM, about 50 mM to about 550 mM, about 100 mMto about 600 mM, about 200 mM to about 600 mM, about 300 mM to about 600mM or about 400 mM to about 600 mM salt. In some embodiments, anequilibration buffer comprises about 500 mM salt. In some embodiments,an equilibration buffer comprises a salt selected from the groupconsisting of sodium chloride (NaCl), sodium acetate(NaAcetate,CH₃COONa), ammonium acetate (NH₄Acetate), magnesium chloride(MgCl₂) or sodium sulfate (Na₂SO₄).

In some embodiments, an equilibration buffer comprises 5 mM to 1 Msodium acetate. In some embodiments, an equilibration buffer comprisesabout 10 mM to about 950 mM, about 10 mM to about 900 mM, about 10 mM toabout 850 mM, about 10M to about 800 mM, 10 mM to about 750 mM, about 10mM to about 700 mM, about 10 mM to about 650 mM, about 10 mM to about600 mM, about 10 mM to about 550 mM, about 50 mM to about 750 mM, about50 mM to about 700 mM, about 50 mM to about 650 mM, about 50 mM to about600 mM, about 50 mM to about 550 mM, about 100 mM to about 600 mM, about200 mM to about 600 mM, about 300 mM to about 600 mM, or about 400 mM toabout 600 mM sodium acetate. In some embodiments, an equilibrationbuffer comprises about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM,about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about400 mM, about 450 mM, about 500 mM, about 550 mM or about 600 mM sodiumacetate. In some embodiments, an equilibration buffer comprises about500 mM sodium acetate.

In some embodiments, an equilibration buffer comprises an amino acid,e.g., histidine, arginine, glycine or citrulline. In some embodiments,an equilibration buffer comprises about 50 mM, about 75 mM, about 100mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225mM, about 250 mM, about 275 mM or about 300 mM of an amino acid (e.g.,histidine, arginine, glycine or citrulline).

In some embodiments, an equilibration buffer comprises an amino acid,e.g., histidine or arginine. In some embodiments, an equilibrationbuffer comprises 100 mM to 300 mM of an amino acid (e.g., histidinearginine, glycine or citrulline). In some embodiments, an equilibrationbuffer comprises about 10 mM to about 600 mM, about 10 mM to about 550mM, about 10 mM to about 500 mM, about 10 mM to about 450 mM about 10 mMto about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300mM, about 50 mM to about 600 mM, about 50 mM to about 550 mM, about 50mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about400 mM, about 50 mM to about 350 mM, about 50 mM, to about 300 mM, about100 mM to about 600 mM, about 100 mM to about 500 mM, about 100 mM toabout 400 mM, about 100 mM to about 300 mM salt, or about 150 mM toabout 250 mM of an amino acid (e.g., histidine). In some embodiments, anequilibration buffer comprises about 200 mM histidine.

In some embodiments, an equilibration buffer comprises a detergent,e.g., P188, Triton X-100, Polysorbate 80, Brij-35 or NP-40. In someembodiments, an equilibration buffer comprises 0.005% to 1.0% of adetergent (e.g., P188). In some embodiments, an equilibration buffercomprises 0.005% to 0.015% of a detergent (e.g., P188). In someembodiments, an equilibration buffer comprises 0.1% to 1.0% of adetergent (e.g., P188). In some embodiments, an equilibration buffercomprises about 0.005% to about 1.0%, about 0.005% to about 0.5%, about0.005% to about 0.1% about 0.005% to about 0.05%, about 0.007% to about0.07%, 0.008% to about 0.05% or about 0.008% to about 0.03% of P188. Insome embodiments, an equilibration buffer comprises about 0.01% to about1.5%, about 0.01% to about 1.0%. about 0.01% to about 0.75%, about 0.05%to about 1.5%, about 0.05% to about 1.0%, about 0.05% to about 0.75%,about 0.1% to about 1.5%, about 0.1% to about 1.0%, about 0.1% to about0.75%, or about 0.25% to about 0.75% P188.

In some embodiments, an equilibration buffer comprises about 0.005%,about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%,about 0.02%, about 0.03% about 0.04%, about 0.05%, about 0.06%, about0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%,about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.5%, about0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%,about 0.85%, about 0.9%, 0.95% or about 1.0% of a detergent (e.g.,P188). In some embodiments, an equilibration buffer comprises about0.01% P188. In some embodiments, an equilibration buffer comprises about0.5% P188.

In some embodiments, an equilibration buffer has a pH of 8 to 10. Insome embodiments, an equilibration buffer has a pH of 8.7 to 9.3. Insome embodiments, an equilibration buffer has a pH of 8.7 to 9.0. Insome embodiments, an equilibration buffer has a pH of about 8.0, about8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7,about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about9.4, about 9.5 or about 10.0. In some embodiments, an equilibrationbuffer has a pH of about 8.8. In some embodiments, an equilibrationbuffer has a pH of about 8.9. In some embodiments, an equilibrationbuffer has a pH of about 9.0.

In some embodiments, an equilibration buffer comprises 20 mM Tris, pH9.0. In some embodiments, an equilibration buffer comprises 100 mM Tris,pH 9.

In some embodiments, an equilibration buffer comprises 20 mM Tris and500 mM NaCl, pH 9.0+/−0.3. In some embodiments, an equilibration buffercomprises 20 mM Tris and 500 mM NH₄Acetate, pH 9.0+/−0.3. In someembodiments, an equilibration buffer comprises about 20 mM Tris, 500 mMsodium acetate, pH 9.0+/−0.3. In some embodiments, an equilibrationbuffer comprises 20 mM Tris and 500 mM Na₂SO₄, pH 9.0+/−0.3.

In some embodiments, an equilibration buffer comprises 20 mM Tris, 7 mMsalt (e.g., NaCl, sodium acetate, ammonium acetate (NH₄Acetate), MgCl₂and Na₂SO₄) pH 9.0. In some embodiments, an equilibration buffercomprises 20 mM Tris, 7 mM sodium acetate, pH 9.0. In some embodiments,an equilibration buffer comprises 20 mM Tris, 14 mM sodium acetate, pH9.0. In some embodiments, an equilibration buffer comprises 20 mM Tris,21 mM sodium acetate, pH 9.0. In some embodiments, an equilibrationbuffer comprises 20 mM Tris, 42 mM sodium acetate, pH 9.0. In someembodiments, an equilibration buffer comprises 20 mM Tris, 49 mM sodiumacetate, pH 9.0. In some embodiments, an equilibration buffer comprises20 mM Tris, 57 mM sodium acetate, pH 9.0. In some embodiments, anequilibration buffer comprises 20 mM Tris, 67 mM sodium acetate, pH 9.0.

In some embodiments, an equilibration buffer comprises 50 mM to 150 mM(e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9). In someembodiments, an equilibration comprises 50 mM to 150 mM (e.g., about 100mM) Tris, 400 mM to 600 mM (e.g., about 500) mM sodium acetate, 0.005%to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9). Insome embodiments, an equilibration buffer comprises 100 mM to 300 mMhistidine (e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g., about200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g.,about 8.8). In some embodiments, an equilibration buffer comprises 50 mMto 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9).

In some embodiments, an equilibration buffer (e.g., a firstequilibration buffer) comprises 100 mM Tris, pH 9. In some embodiments,an equilibration buffer (e.g., a first or a second equilibration buffer)comprises 100 mM Tris, 500 mM sodium acetate, 0.01% P188, pH 8.9. Insome embodiments, an equilibration buffer (e.g., a second or a thirdequilibration buffer) comprises 200 mM histidine, 200 mM Tris, 0.5%P188, pH 8.8. In some embodiments, an equilibration buffer (e.g., athird or a fourth equilibration buffer) comprises 100 mM Tris, 0.01%P188, pH 8.9.

In some embodiments, an equilibration buffer described above may be afirst, second, third and fourth equilibration buffer. In someembodiments, a first, second, third or fourth equilibration buffer isapplied to a column stationary phase in sequential order. In someembodiments, a solution (e.g., an affinity eluate) is applied to thecolumn between application of two equilibration buffers. For example, afirst, second and third equilibration buffer may be applied to a column,followed by application of an affinity eluate, which is followed byapplication of a fourth equilibration buffer. In another example, afirst and second equilibration buffer are applied to a column, followedby application of an affinity eluate, which is followed by applicationof a third equilibration buffer.

In some embodiments, an amount of equilibration buffer applied to acolumn is 1 CV to 5 CV, 4 CV to 6 CV, 4 CV to 10 CV, 4 CV to 15 CV, 4 CVto 21 CV, 10 CV to 21 CV, 15 CV to 21 CV or 19 CV to 21 CV. In someembodiment, an amount of equilibration buffer applied to a column is≥4.5 CV. In some embodiments, an amount of an equilibration bufferapplied to a column is 4.5 CV to 5.5 CV. In some embodiments, an amountof equilibration buffer applied to a column is about 2 CV, about 5 CV orabout 10 CV. In some embodiments, an amount of equilibration bufferapplied to a column is about 5 CV. In some embodiments, an amount ofequilibration buffer applied to a column is about 20 CV.

A solution, including but not limited to an equilibration buffer,applied to a column is set to flow through the stationary phase at aparticular rate (e.g., cm/hr, mL/min) so that the solution within thecolumn is in contact with the stationary phase, for a particular periodof time (referred to herein as “residence time” or “contact time”). Insome embodiments, a residence time of a solution in a column is 0.1min/CV to 10 min/CV, e.g., 0.1 min/CV to 1 min/CV, 1 min/CV to 2 min/CV,2 min/CV to 4 min/CV, 4 min/CV to 6 min/CV, 6 min/CV to 8 min/CV, or 8min/CV to 10 min/CV. In some embodiments, a residence time of a solutionin a column is 0.1 min/CV, about 0.5 min/CV, about 1.5 min/CV, about 2min/CV, about 3 min/CV, about 3.6 min/CV or about 4 min/CV, about 5min/CV, about 6 min/CV, about 7 min/CV, about 8 min/CV, about 9 min/CVor about 10 min/CV. In some embodiments, a residence time of a solutionin a column is 1.5 to 4.5 min/CV. In some embodiments, a residence timeof a solution in a column is 3.5 to 4.5 min/CV.

In some embodiments, a residence time of a solution in a column with aheight of about 5 cm, a diameter of about 0.5 cm and a volume of about1.0 mL is about 0.5 min/CV. In some embodiments, a residence time of asolution in a column with a height of about 15 cm, a diameter of about0.66 cm and a volume of about 5.1 mL is about 0.5 min/CV, about 1.5min/CV or about 4 min/CV. In some embodiments, a residence time of asolution in a column with a height of about 19.5 cm, a diameter of about0.66 and a volume of about 6.67 mL is about 4 min/CV. In someembodiments, a residence time of a solution in a column with a height ofabout 10 cm, a diameter of about 2.5 cm and volume of about 49 mL is 1.5min/CV to 2.5 min/CV (e.g., about 2 min/CV). In some embodiments, aresidence time of a solution in a column with a height of about 16 cm, adiameter of about 10 cm and volume of about 1.256 L to a 1.3 L is 3.5min/CV to 4.5 min/CV (e.g., about 4 min/CV). In some embodiments, aresidence time of a solution in a column with a height of about 20.5 cm,a diameter of 20 cm and a volume of about 6.4 L is about 3.6 min/CV. Insome embodiments, a residence time of a solution in an about 6.4 Lcolumn is 3.5 min/CV to 4.5 min/CV (e.g., about 4 min/CV). In someembodiments, a residence time of a solution, including but not limitedto an equilibration buffer, in a 6.0 L to 6.6 L (e.g., 6.4 L) columncomprising an AEX stationary phase is 3.5 min/CV to 4.5 min/CV (e.g.,about 4 min/CV).

One of ordinary skill will understand that linear velocity (alsoreferred to herein as “linear flow velocity” or “velocity”) of asolution through a column is related, at least in part, to a volume andor dimension of the column and the stationary phase therein. In someembodiments, a linear velocity of a solution, including but not limitedto an equilibration buffer, through a stationary phase in a column is100 cm/hr to 1800 cm/hr, e.g., 100 cm/hr to 200 cm/hr, 200 cm/hr to 400cm/hr, 400 cm/hr to 600 cm/hr, 600 cm/hr to 800 cm/hr, 800 cm/hr to 1000cm/hr, 1000 cm/hr to 1500 cm/hr, or 1500 cm/hr to 1800 cm/hr. In someembodiments, a linear velocity of a solution through a stationary phasein a column is about 100 cm/hr, about 240 cm/hr, about 298 cm/hr, about300 cm/hr, about 600 cm/hr, about 611 cm/hr or about 1790 cm/hr.

In some embodiments, a linear velocity of a solution through astationary phase in a column that is about 5 cm high with a diameter ofabout 0.5 cm and a volume of about 1.0 mL is about 611 cm/hr. In someembodiments, a linear velocity of a solution through a stationary phasein a column that is about 15 cm high with a diameter of about 0.66 cmand a volume of about 5.1 mL is about 600 cm/hr. In some embodiments, alinear velocity of a solution through a stationary phase in a columnthat is about 15 cm high with a diameter of about 0.66 cm and a volumeof about 5.1 mL is about 1790 cm/hr. In some embodiments, a linearvelocity of a solution through a stationary phase in a column that isabout 10 cm high with a diameter of about 2.5 cm and a volume of about49 mL is about 298 cm/hr. In some embodiments, a linear velocity of asolution through a stationary phase in a column that is about 16 cm highwith a diameter of about 10 cm and a volume of about 1256 mL is about240 cm/hr. In some embodiments, a linear velocity of a solution througha stationary phase in a column that is about 20.5 cm high with adiameter of about 20 cm and a volume of about 6.4 L is 270 cm/hr to 330cm/hr (e.g., 300 cm/hr). In some embodiments, a linear velocity of asolution, including but not limited to an equilibration buffer, throughAEX stationary phase in a 6.0 L to 6.6 L (e.g., 6.4 L) column is about270 cm/hr to 330 cm/hr (e.g., about 300 cm/hr).

In some embodiments, a flow rate (i.e., a volumetric flow rate) of asolution, including but not limited to an equilibration buffer, througha stationary phase in a column is 1.0 mL/min to 3.0 L/min, e.g., 1.0mL/min to 10 mL/min, 10 mL/min to 100 mL/min, 100 mL/min to 500 mL/min,500 mL/min to 1000 mL/min, 1 mL/min to 1.5 L/min, 1 mL/min to 2 L/min or2 mL/min to 3 L/min. In some embodiments, a flow rate of a solutionthrough a stationary phase in a column is about 1 mL/min, about 1.28mL/min, about 1.67 mL/min, about 314 mL/min, about 1.57 L/min, about 1.8L/min, about 2 L/min, about 3 L/min.

In some embodiments, a flow rate of a solution through a stationaryphase in a column with a height of about 15 cm, a diameter of about 0.66and volume of about 5.1 mL is about 1.28 mL/min. In some embodiments, aflow rate of solution through a stationary phase in a column with aheight of about 19.5 cm, a diameter of about 0.66 and volume of about6.67 mL is about 1.67 mL/min. In some embodiments, a flow rate of asolution through a stationary phase in a column with a height of about16 cm, a diameter of 10 cm and a volume of about 1256 mL is about 314mL/min. In some embodiments, a flow rate of a solution through astationary phase in a column with a height of about 20.5 cm, a diameterof about 20 cm and a volume of about 6.4 L is about 1.8 L/min. In someembodiments, a flow rate of a solution, including but not limited to anequilibration buffer, through an AEX stationary phase in a 6.0 L to 6.6L (e.g., 6.4 L) column is 1.5 mL/min to 2.0 L/min (e.g., about 1.8L/min).

A method of preparing an AEX stationary phase for use in a method ofpurifying a rAAV (e.g., rAAV9, rAAV3B or others) vector from a solution(e.g., an affinity eluate) by AEX comprises equilibrating the AEXstationary phase in a column. In some embodiments, equilibratingprecedes loading a solution comprising a rAAV vector to be purified ontoa column. In some embodiments, equilibrating follows loading a solutioncomprising a rAAV vector to be purified onto a column.

In some embodiments, equilibrating comprises application of anequilibration buffer comprising 50 mM to 150 mM (e.g., about 100 mM)Tris, pH 8.5 to 9.5 to an AEX stationary phase in a column. In someembodiments, equilibrating comprises application of 4.5 CV to 5.5 CV(e.g., about 5 CV) of an equilibration buffer comprising 100 mM Tris, pH9 to a 6.0 L to 6.6 L (e.g., 6.4 L) column comprising AEX stationaryphase at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 300cm/hr), a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min)and/or a residence time of 3.5 min/CV to 4.5 min/CV (e.g., about 4min/CV).

In some embodiments, equilibrating comprises application of anequilibration buffer comprising 400 mM to 600 mM sodium acetate, 50 mMto 150 mM Tris and 0.005% to 0.015% P188, pH 8.5 to 9.5 to an AEXstationary phase in a column. In some embodiments, equilibrationcomprises application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of anequilibration buffer comprising 100 mM Tris, 500 mM sodium acetate,0.01% P188, pH 8.9 to a column comprising an AEX stationary phase at alinear velocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr, about300 cm/hr), a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8L/min) and/or a residence time of 1.5 min/CV to 4.5 min/CV (e.g., about2 min/CV, about 4 min/CV). In some embodiments, a column is 6.0 L to 6.6L (e.g., 6.4 L). In some embodiments, a column is 30 mL to 70 mL (e.g.,about 49 mL, about 52 mL).

In some embodiments, equilibrating comprises application of anequilibration buffer comprising 100 mM to 300 mM histidine, 100 mM to300 mM Tris, and 0.0% to 1.0% P188, pH 8.5 to 9.5 to an AEX stationaryphase in a column. In some embodiments, equilibrating comprisesapplication of ≥4.5 CV (e.g., about 5 CV) of an equilibration buffercomprising 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.8 to a columncomprising an AEX stationary phase at a linear velocity of 270 cm/hr to330 cm/hr (e.g., about 298 cm/hr, about 300 cm/hr), a flow rate of 1.5L/min to 2.0 L/min (1.8 L/min) and/or a residence time of 1.5 min/CV to4.5 min/CV (e.g., about 2 min/CV, about 4 min/CV). In some embodiments,a column is 6.0 L to 6.6 L (e.g., 6.4 L). In some embodiments, a columnis 30 mL to 70 mL (e.g., about 49 mL, about 52 mL).

In some embodiments, equilibration comprises application of anequilibration buffer comprising 50 mM to 150 mM Tris and 0.005% to0.015% P188, pH 8.5 to 9.5 to an AEX stationary phase in a column. Insome embodiments, equilibration comprises application of 4.5 CV to 5.5CV (e.g., about 5 CV) of an equilibration buffer comprising 100 mM Tris,0.01% P188, pH 8.9 to a column comprising an AEX stationary phase at alinear velocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr, about300 cm/hr), a flow rate of 1.5 L/min to 2.0 L/min (1.8 L/min) and/or aresidence time of 1.5 min/CV to 4.5 min/CV (e.g., 2 min/CV, 4 min/CV).In some embodiments, a column is 6.0 L to 6.6 L (e.g., 6.4 L). In someembodiments, a column is 30 mL to 70 mL (e.g., about 49 mL, about 52mL).

In some embodiments, the present disclosure provides a method preparingan AEX stationary phase for use in a method of purifying a rAAV (e.g.,rAAV9, rAAV3B or others) vector by AEX, the method comprising a step of:i) pre-use flushing comprising application of ≥4.5 CV (e.g., about 5 CV)of water for injection to the AEX stationary phase in a column; ii)sanitizing comprising application of about 5 CV to 10 CV (e.g., about 8CV) or about 14.4 to 17.6 CV (e.g., about 16 CV) of a solutioncomprising 0.1 M to 1.0 M (e.g., about 0.5 M) NaOH to the AEX stationaryphase in the column, optionally, by upward flow; iii) regeneratingcomprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of asolution comprising 1 M to 3 M (e.g., about 2 M) NaCl, 50 mM to 150 mM(e.g., about 100 mM Tris), pH 8.5 to 9.5 (e.g., about 9) to the AEXstationary phase in the column; iv) equilibration comprising applicationof 4.5 CV to 5.5 CV (e.g., about 5 CV) of a solution comprising 50 mM to150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to theAEX stationary phase in the column; v) equilibration comprisingapplication of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibrationbuffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 400 mM to600 mM (e.g., about 500 mM) sodium acetate, 0.005% to 0.015% (e.g.,about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) to the AEX stationaryphase in the column; vi) equilibration comprising application of ≥4.5 CV(e.g., about 5 CV) of an equilibration buffer comprising 100 mM to 300mM (e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g., about 200 mM)Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., 8.8) tothe AEX stationary phase in the column; and/or vii) equilibrationcomprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of anequilibration buffer comprising 50 mM to 150 mM (e.g., about 100 mM)Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g.,8.9) to the AEX stationary phase in the column; optionally wherein atleast one of steps i)-vii) is performed at a linear velocity of 270cm/hr to 330 cm/hr (e.g., about 298 cm/hr, about 300 cm/hr), and/or aresidence time of 1.5 min/CV to 4.5 min/CV (e.g., about 2 min/CV, about4 min/CV); optionally wherein the AEX stationary phase is POROS™ 50 HQ;optionally wherein the rAAV vector is a rAAV9 vector or a rAAV3B vector,and optionally wherein a step (e.g., a load step) may occur between anystep of equilibration. In some embodiments, at least one of stepsi)-vii) is performed at a flow rate of 1.5 L/min to 2.0 L/min (e.g.,about 1.8 L/min) through a 6 L to 6.6 L column (e.g., about 6.4 L), orabout 314 mL/min through a 1.3 L column. One of ordinary skill willunderstand that the order of the above steps may be varied.

Dilution and Filtration

A method of purifying a rAAV (e.g., rAAV9, rAAV3B or others) vector froma solution (e.g., an affinity eluate) by AEX comprises preparation ofthe solution by diluting, and optionally filtering, the solution. Asolution comprising a rAAV vector to be purified may be an affinityeluate, a supernatant from a cell lysate and/or a post-harvest solutionhaving undergone at least one purification or processing step. Asolution comprising a rAAV vector to be purified may be diluted, andoptionally filtered prior to loading onto an AEX column in order to makethe solution compatible with processing through the AEX column. In someembodiments, diluting, and optionally filtering, a solution comprising arAAV vector to be purified results in a change in pH and/or conductivityof the solution. In some embodiments, a solution comprising a rAAVvector to be purified is an eluate resulting from affinitychromatography purification of a rAAV vector produced in a 1 L to 2000 L(or greater) single use bioreactor (SUB).

A method of preparing a solution comprising a rAAV vector forpurification by AEX comprises i) diluting an affinity eluate, andoptionally ii) filtering the affinity eluate from step i) to produce thediluted affinity eluate (also referred to herein as a “diluted affinitypool,” “load,” or “AEX load”). In some embodiments, pH of an affinityeluate after dilution, and optional filtration is increased as comparedto pH of the affinity eluate before the dilution. In some embodiments,conductivity of an affinity eluate after dilution, and optionalfiltration is decreased as compared to conductivity of the affinityeluate before the dilution. In some embodiments, the diluted, andoptionally filtered affinity eluate is loaded on an AEX stationaryphase.

In some embodiments, an affinity eluate is generated from affinitypurification of a rAAV vector produced in a vessel (e.g., a single usebioreactor (SUB)) with a volume of 1 mL to 2000 L, or greater than 2000L. In some embodiments, an affinity eluate is generated from affinitychromatography purification of a rAAV vector produced in a vessel (e.g.,a SUB) with a volume of about 1 mL, about 10 mL, about 50 mL, about 100mL, about 250 mL, about 500 mL, about 750 mL, about 1 L, about 50 L,about 100 L, about 250 L, about 500 L, about 1000 L, about 2000 L orgreater. In some embodiments, an affinity eluate is generated fromaffinity purification of a rAAV vector produced in a vessel (e.g., aSUB) with a volume of 1 mL to 100 mL, 100 mL to 500 mL, 500 mL to 750mL, 750 mL, to 1 L, 1 L to 10 L, 10 L to 50 L, 50 L to 100 L, 100 L to250 L, 250 L to 500 L, 500 L to 750 L, 750 L to 1000 L, 1000 L to 1500L, 1500 L to 2000 L, 2000 L to 3500 L, 3500 L to 4000 L or 4500 L to5000 L. In some embodiments, an affinity eluate is generated fromaffinity purification of a rAAV vector produced in a vessel (e.g., aSUB) with a volume of 1 mL to 5000 L, 100 mL to 5000 L, 100 mL to 4000L, 100 mL to 2000 L, 100 mL to 1000 L, 1 L to 5000 L, 1 L to 4000 L, 1Lto 2000 L, 1 L to 1000 L, 500 mL to 5000 L, 500 mL to 2000 L or 500 mLto 1000 L.

In some embodiments, diluting a solution comprising a rAAV vector to bepurified (e.g., an affinity eluate) comprises diluting the solutionabout 2 to 25-fold or about 5 to 20-fold, or about 10 to 20-fold (e.g.,about 5-fold, about, 6-fold, about 7-fold, about 8-fold, about 9-fold,about 10-fold, about 11-fold, about 12-fold, about 13-fold, about14-fold, about 15-fold, about 20-fold, about 25-fold) to produce adiluted affinity eluate. In some embodiments, diluting a solutioncomprising a rAAV vector to be purified (e.g., an affinity eluate)comprises diluting the solution about 2-fold. In some embodiments,diluting a solution comprising a rAAV vector to be purified (e.g., anaffinity eluate) comprises diluting the solution about 15-fold.

In some embodiments, diluting a solution comprising a rAAV vector to bepurified (e.g., an affinity eluate) is performed “in-line” with thecolumn, and wherein a dilution solution (diluent) is delivered through afirst tubing to a Y-connector, and the solution comprising a rAAV vectorto be purified is delivered through a second tubing to the Y-connector,and optionally wherein a static mixer is contained within a third tubinglocated after the Y-connector.

In some embodiments, diluting a solution comprising a rAAV vector to bepurified (e.g., an affinity eluate) is performed “in-line” and directedinto a holding vessel (e.g., a break tank). For example, a dilutionsolution (diluent) is delivered through a first tubing to a Y-connector,and a solution comprising a rAAV vector to be purified is deliveredthrough a second tubing to the Y-connector, wherein the end of theY-connector is connected to a holding vessel which is optionally,connected to a chromatography column (e.g., an AEX column).

In some embodiments, diluting comprises delivery of a dilution solutionthrough a first tubing to a Y-connector at a flow rate of 1 to 5 mL/min(e.g., about 3.5 mL/min) and delivery of a solution comprising a rAAVvector to be purified (e.g., an affinity eluate) through a second tubingat a flow rate of 0.1 to 2 mL/min (e.g., about 0.25 mL/min).

In some embodiments, diluting comprises delivery of a dilution solutionthrough a first tubing to a Y-connector at a flow rate of about 3.5mL/min and delivery of an affinity eluate through a second tubing at aflow rate of about 0.25 mL/min, such that the affinity eluate is dilutedabout 15-fold.

In some embodiments, diluting comprises dilution of a solutioncomprising a rAAV vector to be purified (e.g., an affinity eluate) witha dilution solution comprising a buffering agent (Tris, BIS-Trispropane, diethanolamine, diethylamine, tricine, triethanolamine and/orbicine). In some embodiments, a solution comprising a rAAV vector to bepurified (e.g., an affinity eluate) is diluted with a dilution solutioncomprising 10 mM to 500 mM buffering agent (e.g., Tris). In someembodiments, a dilution solution comprises about 10 mM to about 450 mM,about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM toabout 300 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM,about 50 mM, about 350 mM, about 50 mM to about 300 mM, about 100 mM toabout 450 mM, about 100 mM to about 400 mM, about 100 mM to about 350mM, about 100 mM to about 300 mM, or about 150 mM to about 250 mM Tris.In some embodiments, a dilution solution comprises about 200 mM Tris.

In some embodiments, a dilution solution comprises an amino acid, e.g.,histidine, arginine, glycine or citrulline. In some embodiments, adilution solution comprises about 50 mM, about 75 mM, about 100 mM,about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM,about 250 mM, about 275 mM, about 300 mM, about 350 mM, about 400 mM,about 450 mM, about 500 mM, about 550 mM or about 600 mM of an aminoacid (e.g., histidine, arginine, glycine or citrulline).

In some embodiments, dilution solution comprises an amino acid, e.g.,histidine or arginine. In some embodiments, an dilution solutioncomprises 10 mM to 600 mM of an amino acid (e.g., histidine arginine,glycine or citrulline). In some embodiments, an equilibration buffercomprises about 10 mM to about 600 mM, about 10 mM to about 550 mM,about 10 mM to about 500 mM, about 10 mM to about 450 mM about 10 mM toabout 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM,about 50 mM to about 600 mM, about 50 mM to about 550 mM, about 50 mM toabout 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM,about 50 mM to about 350 mM, about 50 mM, to about 300 mM, about 100 mMto about 600 mM, about 100 mM to about 500 mM, about 100 mM to about 400mM, about 100 mM to about 300 mM, or about 150 mM to about 250 mM of anamino acid (e.g., histidine). In some embodiments, a dilution solutioncomprises about 200 mM histidine.

In some embodiments, a dilution solution comprises a detergent, e.g.,P188, Triton X-100, Polysorbate 80, Brij-35 or NP-40. In someembodiments, dilution solution comprises 0.005% to 1.5% detergent (e.g.,P188). In some embodiments, a dilution solution comprises 0.1% to 1.0%detergent (e.g., P188). In some embodiments, a dilution solutioncomprises about 0.01% to about 1.5%, about 0.01% to about 1.0%. about0.01% to about 0.75%, about 0.05% to about 1.5%, about 0.05% to about1.0%, about 0.05% to about 0.75%, about 0.1% to about 1.5%, about 0.1%to about 1.0%, about 0.1% to about 0.75%, or about 0.25% to about 0.75%detergent (e.g., P188). In some embodiments, a dilution solutioncomprises about 0.5% P188.

In some embodiments, a dilution solution has a pH of 8 to 10. In someembodiments, a dilution solution has a pH of 8.5 to 9.5. In someembodiments, a dilution solution has a pH of 8.7 to 9.0. In someembodiments, a dilution solution has a pH of about 8.0, about 8.1, about8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8,about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about9.5 or about 10.0. In some embodiments, a dilution solution has a pH ofabout 8.8. In some embodiments, a dilution solution has a pH of about8.9. In some embodiments, a dilution solution has a pH of about 9.0.

In some embodiments, diluting comprises dilution of a solutioncomprising a rAAV vector to be purified (e.g., an affinity eluate) witha buffer selected from the group consisting of 20 mM Tris, pH 9; 1 MTris Base, pH 11; 100 mM Tris, pH 9; 100 mM Tris, 0.01% P188, pH 9; 100mM Tris, 0.1% P188, pH 9; 100 mM Tris, 1.0% P188, pH 9; 1 M Tris, pH 9;150 mM acetate, 100 mM glycine, 25 mM MgCl₂, pH 4.2; 5 mM Arginine, 2 mMMgCl₂, 0.1% P188, 100 mM Tris, pH 8.9; 50 mM arginine, 2 mM MgCl₂, 0.1%P188, 100 mM Tris, pH 9; 500 mM Arginine, 2 mM MgCl₂. 0.1% P188, 400 mMTris, pH 9.1; 200 mM Glycine, 5 mM MgCl₂, 200 mM Tris, pH 8.9; 200 mMHistidine. 200 mM Tris, pH 8.9; 200 mM Histidine, 200 mM Tris, 5 mMMgCl₂, pH 8.9; 200 mM Histidine, 200 mM Tris, 5 mM MgCl₂, 5% Glycerol,pH 8.9; 200 mM Histidine, 250 mM Tris, 10 mM MgCl₂, 25% Glycerol, pH8.9; 200 mM Histidine, 200 mM Tris, 5 mM MgCl₂, 5% Iodixanol pH 8.8; 200mM Histidine, 200 mM Tris, 10 mM MgCl₂, 25% Iodixanol, pH 8.8; 200 mMHistidine, 200 mM Tris, 0.5% Triton X-100, pH 8.9; 200 mM histidine, 200mM Tris, 0.5% P188, pH 8.8; and a combination thereof.

In some embodiments, diluting comprises dilution of a solutioncomprising a rAAV vector to be purified (e.g., an affinity eluate) witha buffer comprising about 20 mM Tris, pH 9, about 1 M Tris base, pH 11,or both. In some embodiments, diluting comprises dilution of a solutioncomprising a rAAV vector to be purified (e.g., an affinity eluate) 7 to8 fold (e.g., about 7.1 fold) with a buffer comprising about 20 mM Tris,pH 9, about 1 M Tris Base, pH 11, or both.

In some embodiments, diluting comprises dilution of a solutioncomprising a rAAV vector to be purified (e.g., an affinity eluate) witha buffer comprising 100 mM to 300 mM (e.g., about 200 mM) histidine, 100mM to 300 mM (e.g., about 200 mM) Tris, 0.1% to 1.0% (e.g., 0.5%) P188,pH 8.7 to 9.0. In some embodiments, diluting comprises dilution of asolution comprising a rAAV vector to be purified (e.g., an affinityeluate) 10 to 20 fold by weight (e.g., about 15 fold) with a buffercomprising about 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.7 to 9.0(e.g., about 8.8). In some embodiments, diluting comprises dilution ofan affinity eluate comprising a rAAV vector to be purified 14.4 to 15.5fold by weight (e.g., about 15 fold) with a buffer comprising about 200mM histidine, 200 mM Tris, 0.5% P188, pH 8.7 to 9.0 (e.g., about pH8.8), and thereby forming a diluted affinity eluate.

In some embodiments, prior to dilution of a solution comprising a rAAVvector to be purified (e.g., an affinity eluate) the solution is spikedwith 20 mM MgCl₂ so that the concentration of MgCl₂ in the dilutedsolution is about 1.7 mM. In some embodiments, MgCl₂ stabilizes the rAAVvectors in a solution.

In some embodiments, filtering comprises filtration of a solutioncomprising a rAAV vector to be purified (e.g., an affinity eluate, adiluted affinity eluate) prior to loading the solution onto an AEXcolumn. In some embodiments, prior filtering, a filter is pre-wet withwater for injection and/or a dilution solution. In some embodiments,filtering comprises filtration of a solution comprising a rAAV vector tobe purified (e.g., an affinity eluate, a diluted affinity eluate)through a filter which collects aggregates, such as nucleic acid orprotein aggregates, or other high molecular mass species, but allows AAVcapsids to flow through. In some embodiments, a filter is an 0.1 μm to0.45 μm filter (e.g., a 0.2 μm polyethersulfone (PES) filter or a 0.45μm PES filter). In some embodiments, filtering comprises filtration of adiluted affinity eluate comprising a rAAV vector to be purified throughan 0.2 μm filter prior to loading onto an AEX column. A filter used tofilter a solution comprising a rAAV vector to be purified (e.g., anaffinity eluate, a diluted affinity eluate) may be separate from thecolumn, or may be in-line with the column or chromatography apparatus(also referred to as a chromatography skid).

In some embodiments, filtering comprises filtration of a dilutedaffinity eluate comprising a rAAV vector to be purified through anin-line 0.2 μm filter before loading the eluate onto an AEX column.

In some embodiments, pH of a solution comprising a rAAV vector to bepurified (e.g., an affinity eluate) is 3.0 to 4.4 prior to diluting, andoptionally filtering, and pH of the solution comprising a rAAV vector tobe purified (e.g., an affinity eluate) after diluting, and optionallyfiltering, is 8.5 to 9.5, 8.7 to 9.0 or ≥8.6 (e.g., about pH 8.8, pH9.0).

In some embodiments, conductivity of a solution comprising a rAAV vectorto be purified (e.g., an affinity eluate) is 5.0 mS/cm to 7.0 mS/cm(e.g., about 5.5 mS/cm to 6.5 mS/cm) prior to diluting, and optionallyfiltering, and conductivity of the solution comprising a rAAV vector tobe purified (e.g., an affinity eluate) after diluting, and optionallyfiltering, is 1.7 mS/cm to 3.5 mS/cm, 1.8 mS/cm to 2.8 mS/cm, 2.2 mS/cmto 2.6 mS/cm or ≤2.5 mS/cm. In some embodiments, conductivity of anaffinity eluate after diluting, and optionally filtering, is about 1.8mS/cm to about 2.8 mS/cm. In some embodiments, conductivity of anaffinity eluate following dilution, and optionally filtering, is about2.3+/−0.5 mS/cm.

As used herein, the term “percent VG dilution yield” or “% VG dilutionyield” refers to the amount of VG present in a diluted affinity pool(also referred to herein as a diluted affinity eluate) as a percentageof the amount of VG present in the affinity pool (also referred toherein as an affinity eluate) prior to dilution. For instance, % VGdilution yield=((amount of VG in diluted affinity pool)/(amount of VG inaffinity pool))*100.

In some embodiments, a percentage of VG recovered in a diluted, andoptionally filtered solution (% VG dilution yield) comprising a rAAVvector to be purified (e.g., an affinity eluate) is 60% to 100% of theVG present in a solution (e.g., an affinity eluate) prior to diluting,and optionally filtering. In some embodiments, a % VG yield of adiluted, and optionally filtered solution comprising a rAAV vector to bepurified (e.g., an affinity eluate) is 60% to 70%, 70% to 80%, 80% to90%, 90% to 100% of the VG present in a solution (e.g., an affinityeluate) prior to diluting, and optionally filtering. In someembodiments, a % VG yield of a diluted, and optionally filtered solutioncomprising a rAAV vector to be purified (e.g., an affinity eluate) isabout 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, about 98%, about 99% or about 100% of the VG present ina solution prior to diluting, and optionally filtering.

In some embodiments of diluting an affinity eluate according to methodsdisclosed herein results in % VG dilution yield of 88%+/−36%. In someembodiments of diluting an affinity eluate according to methodsdisclosed herein results in % VG dilution yield of 120%+/−12%. Dilutingan affinity eluate resulting from affinity chromatography purificationof a rAAV vector produced in a 250 L SUB results in a % VG dilutionyield of 35% to 100% (e.g., 41% to 92%). Diluting an affinity eluateresulting from affinity chromatography purification of a rAAV vectorproduced in a 2000 L SUB results in a % VG dilution yield of 70%to >100% (e.g., 88% to 154%).

In some embodiments, a Z-average (given in units of nm, and determinedby dynamic light scattering (DLS) of a diluted and, optionally filteredsolution comprising a rAAV vector to be purified (e.g., an affinityeluate) is measured. A Z-average measures the level of aggregation ofrAAV capsids present in a solution. In some embodiments, a Z-average ofa diluted and, optionally filtered solution comprising a rAAV vector tobe purified is about 15 nm to 40 nm, 15 nm to 20 nm, 20 nm to 30 nm or30 nm to 40 nm. In some embodiments, a Z-average of a diluted and,optionally filtered solution comprising a rAAV vector to be purified isabout 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about20 nm, 21 nm, about 22 nm, about 23 nm, about 24 nm, about 25 nm, about26 nm, about 27 nm, about 28 nm, about 29 nm, about 30 nm, about 35 nmor about 40 nm.

A method of purifying a rAAV (e.g., rAAV9, rAAV3B or others) vector froma solution (e.g., an affinity eluate) by AEX comprises diluting thesolution by 14 to 16 fold (e.g., about 15 fold) with a buffer comprising100 mM to 300 mM (e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g.,about 200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.7 to 9.0(e.g., about pH 8.8); and optionally comprises filtering comprisingfiltration of the diluted solution through a 0.1 μm to 0.45 μm (e.g.,about 0.2 μm) filter, and wherein the diluted, and optionally filteredsolution has a pH of about 8.6 to 9.0 (e.g., about pH 8.9) and aconductivity of 1.8 mS/cm to 2.8 mS/cm.

A method of preparing an affinity eluate comprising a rAAV vector forpurification by AEX chromatography, as disclosed herein, comprises i)diluting the affinity eluate 2 to 25-fold (e.g., about 15-fold) with abuffer comprising 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.8; andii) optionally filtering the affinity eluate from step i) through a 0.2μm filter to produce the diluted affinity eluate; wherein the pH of thediluted affinity eluate is increased as compared to the pH of theaffinity eluate; wherein the conductivity of the diluted affinity eluateis decreased as compared to the conductivity of the affinity eluate;optionally wherein the rAAV vector is an AAV9 vector or an AAV3B vector;and optionally wherein the affinity eluate is produced by affinitypurification of a rAAV vector produced in a vessel (e.g., a SUB) with avolume of 250 L or 2000 L.

Load

A method of purifying a rAAV (e.g., rAAV9, rAAV3B or others) vector byAEX disclosed herein comprises loading a solution comprising a substanceto be purified (e.g., a rAAV vector) onto an AEX stationary phase in acolumn. Loading may be performed by gravity feeding the load onto thecolumn or pumping the load onto the chromatography column. In someembodiments, a solution comprising a rAAV vector to be purified by AEXis selected from the group consisting of an affinity eluate, asupernatant from a cell lysate and a post-harvest solution, each havingundergone at least one other purification or processing step (e.g., celllysis, flocculation, filtration, dilution, pH adjustment,chromatography). A solution comprising a rAAV vector to be purified maybe diluted, filtered and/or pH adjusted prior to loading the solutiononto an AEX column in order to make the solution compatible withprocessing through the AEX column. In some embodiments, a solutioncomprising a rAAV vector to be purified is an eluate resulting fromaffinity chromatography purification of a rAAV vector produced in a 100L to 500 L (e.g., about 250 L), 1000 L to 3000 L (e.g., about 2000 L) orlarger vessel (e.g., a single use bioreactor (SUB)), and wherein theeluate has been diluted and filtered.

In some embodiments, loading comprises application of a diluted, andoptionally filtered solution (e.g., an affinity eluate) comprising about2.0×10¹² vector genomes (VG)/mL to 2.0×10¹⁵ VG/mL, e.g., 2.0×10¹² VG/mLto 2.0×10¹³ VG/mL, 2.0×10¹³ VG/mL to 2.0×10¹⁴ VG/mL, 1.0×10¹⁴ VG/mL to3.0×10¹⁴ VG/mL, 2.0×10¹⁴ VG/mL to 2.0×10¹⁵ VG/mL, or more of columnvolume (also referred to as a “column challenge VG/mL resin”) onto anAEX column, as measured by qPCR analysis of a sequence within the vectorgenome. In some embodiments, loading comprises application of a dilutedsolution (e.g., an affinity eluate) comprising 6.3×10¹³ to 9.4×10¹³VG/mL of column volume onto an about 30 mL to 70 mL AEX column asmeasured by qPCR analysis of a transgene sequence within the vectorgenome (e.g., wherein the transgene is an ATP7B transgene). In someembodiments, loading comprises application of a diluted and, optionallyfiltered solution (e.g., an affinity eluate) comprising 5×10¹³ to1.3×10¹⁴ VG/mL of column volume onto an about 1.3 L AEX column asmeasured by qPCR analysis of ITR sequences within the vector genome. Insome embodiments, loading comprises application of a diluted and,optionally filtered solution (e.g., an affinity eluate) comprising2.6×10¹² to 6.8×10¹³ VG/mL of column volume onto an about 6.4 L AEXcolumn as measured by qPCR analysis of a transgene sequence within thevector genome.

In some embodiments, loading comprises application of a diluted and,optionally filtered solution (e.g., an affinity eluate) comprising2.5×10¹⁵ VG/L to 2.5×10¹⁶ VG/L, 2.5×10¹⁶ VG/L to 2.5×10¹⁷ VG/L, 2.5×10¹⁵VG/L to 3.0×10¹⁷ VG/L or more of column volume onto an AEX column.

In some embodiments, loading comprises application of a diluted and,optionally filtered solution (e.g., an affinity eluate) comprising8.0×10¹² total VG to 2.0×10¹⁸ total VG, e.g., 8.0×10¹² total VG to8.0×10¹³ total VG, 8.0×10¹³ to 8.0×10¹⁴ total VG, 8.0×10¹⁴ total VG to8.0×10¹⁵ total VG, 8.0×10¹⁵ total VG to 8.0×10¹⁶ total VG, 8.0×10¹⁶total VG to 8.0×10¹⁷ total VG, 8.0×10¹⁷ total VG to 2.0×10¹⁸ total VG,or more onto an AEX column. In one embodiment, loading comprisesapplication of a diluted and, optionally filtered solution (e.g., anaffinity eluate) comprising ≤15×10¹⁶ VG/L of column volume onto an AEXcolumn, and optionally wherein the VG are measured by quantitativepolymerase chain reaction (qPCR) analysis of the transgene.

When a solution comprising a rAAV vector to be purified (e.g., anaffinity eluate) is loaded onto a column, the solution flows through thecolumn stationary phase at a particular rate (e.g., cm/hr, mL/min) andis in contact with the stationary phase for a particular period of time(i.e., residence time).

In some embodiments, a residence time of a solution comprising a rAAVvector loaded onto a column is 0.1 min/CV to 5 min/CV, e.g., 0.1 min/CVto 1.0 min/CV, 1.0 min/CV to 2 min/CV, 2 min/CV to 3 min/CV, 3 min/CV to4 min/CV, 4 min/CV to 5 min/CV or more. In some embodiments, a residencetime of a solution comprising a rAAV vector loaded onto a column isabout 0.5 min/CV. In some embodiments, a residence time of a solutioncomprising a rAAV vector loaded onto a column is about 1.5 min/CV. Insome embodiments, a residence time of a solution comprising a rAAVvector loaded onto a column is about 2.0 min/CV. In some embodiments, aresidence time of a solution comprising a rAAV vector loaded onto acolumn is 3.5 min/CV to 4.5 min/CV. In some embodiments, a residencetime of a diluted and/or filtered affinity eluate comprising a rAAVvector loaded on a 6.0 L to 6.6 L (e.g., about 6.4 L) AEX column is 3.0min/CV to 5.0 min/CV (e.g., about 4 min/CV).

In some embodiments, a linear velocity of a solution comprising a rAAVvector loaded onto a column is 100 cm/hr to 1800 cm/hr, e.g., 100 cm/hrto 200 cm/hr, 200 cm/hr to 400 cm/hr, 400 cm/hr to 600 cm/hr, 600 cm/hrto 800 cm/hr, 800 cm/hr to 1000 cm/hr, 1000 cm/hr to 1500 cm/hr, 1500cm/hr to 1800 cm/hr. In some embodiments, a linear velocity of asolution comprising a rAAV vector loaded onto a column is 270 cm/hr to330 cm/hr (e.g. about 298 cm/hr, about 300 cm/hr). In some embodiments,a linear velocity of a solution comprising a rAAV vector loaded onto acolumn is about 300 cm/hr, about 600 cm/hr, about 611 cm/hr or about1790 cm/hr. In some embodiments, a linear velocity of a diluted, andoptionally filtered affinity eluate comprising a rAAV vector loaded on a6.0 L to 6.6 L (e.g., about 6.4 L) AEX column is 270 cm/hr to 330 cm/hr(e.g., about 300 cm/hr).

In some embodiments, a flow rate of a solution comprising a rAAV vectorloaded onto a column is 1.0 mL/min to 3.0 L/min, e.g., 1.0 mL/min to 10mL/min, 10 mL/min to 100 mL/min, 100 mL/min to 500 mL/min, 500 mL/min to1000 mL/min, 1 mL/min to 1.5 L/min, 1 mL/min to 2 L/min, 2 mL/min to 3L/min. In some embodiments, a flow rate of a solution comprising a rAAVvector loaded onto a column is about 1.28 mL/min. In some embodiments, aflow rate of a solution comprising a rAAV vector loaded onto a column isabout 314 mL/min. In some embodiments, a flow rate of a solutioncomprising a rAAV vector through stationary phase in a column is 1.5L/min to 2.0 L/min. In some embodiments, a flow rate of a solutioncomprising a rAAV vector loaded onto a column is about 1.8 L/min. Insome embodiments, a flow rate of a diluted and/or filtered affinityeluate comprising a rAAV vector loaded on a 6.0 L to 6.6 L (e.g., about6.4 L) column is 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min).

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from an affinity eluate comprises i) diluting theaffinity eluate with a buffer comprising a detergent (e.g., P188), anamino acid (e.g., histidine) and a buffer (e.g., Tris); ii) optionallyfiltering the diluted affinity eluate; and iii) loading the diluted, andoptionally filtered affinity eluate onto a column comprising an AEXstationary phase wherein the AEX stationary phase has been flushed,sanitized, rinsed and/or equilibrated prior to loading, and optionallywherein the AEX stationary phase is POROS™ 50 HQ.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from an affinity eluate comprises i) diluting theaffinity eluate 14.4 to 15.5 fold (e.g., about 15 fold) with a buffercomprising about 100 mM to 300 mM (e.g., about 200 mM) histidine, 100 mMto 300 mM (e.g., about 200 mM) Tris, 1.0% to 1.5% (e.g., about 0.5%)P188, pH 8.7 to 9.0; ii) optionally filtering the diluted affinityeluate through an in-line 0.1 to 0.45 μm (e.g., about 0.2 μm) filter;and iii) loading the diluted and filtered affinity eluate onto a columncomprising an AEX stationary phase; optionally wherein at least one stepis performed at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about298 cm/hr, about 300 cm/hr), a flow rate of 1.5 L/min to 2.0 L/min(e.g., about 1.8 L/min) through column and/or a residence time of 1.5min/CV to 4.5 min/CV (e.g., about 2 min/CV, about 4 min/CV) and,optionally wherein the AEX stationary phase is POROS™ 50 HQ. In someembodiments, a column is a 6.0 L to 6.6 L (e.g., about 6.4 L) column,

In some embodiments, the present disclosure provides a method ofpurifying a rAAV (e.g., rAAV9, rAAV3B or others) vector by AEX, themethod comprising a step of: i) pre-use flushing comprising applicationof ≥4.5 CV (e.g., about 5 CV) of water for injection to an AEXstationary phase in a column; ii) sanitizing comprising application of 5CV to 10 CV (e.g., about 8 CV) or 14.4 to 17.6 CV (e.g., about 16 CV) ofa solution comprising 0.1 M to 1.0 M (e.g., about 0.5 M) NaOH to the AEXstationary phase in the column, optionally by upward flow; iii)regenerating comprising application of 4.5 CV to 5.5 CV (e.g., about 5CV) of a solution comprising 1 M to 3 M (e.g., about 2 M) NaCl), 50 mMto 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) tothe AEX stationary phase in the column; iv) equilibration comprisingapplication of 4.5 CV to 5.5 CV (e.g., about 5 CV) of a solutioncomprising 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5(e.g., about 9) to the AEX stationary phase in the column; v)equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5CV) of an equilibration buffer comprising 50 mM to 150 mM (e.g., about100 mM) Tris, 400 mM to 600 mM (e.g., about 500 mM) sodium acetate,0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.0 (e.g., about8.9) to the AEX stationary phase in the column; vi) equilibrationcomprising application of ≥4.5 CV (e.g., about 5 CV) of an equilibrationbuffer comprising 100 mM to 300 mM (e.g., about 200 mM) histidine, 100mM to 300 mM (e.g., about 200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%)P188, pH 8.5 to 95. (e.g., about 8.8) to the AEX stationary phase in thecolumn; vii) loading the affinity eluate to an AEX stationary phase inthe column, optionally wherein the eluate has been a) diluted about 14.4to 15.5 fold (e.g., about 15 fold) with a buffer comprising 100 mM to300 mM (e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g., about 200mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.7 to 9.0, andoptionally b) filtered through an in-line 0.1 μm to 0.45 μm (e.g., about0.2 μm) filter prior to application to the stationary phase; and/orviii) equilibration comprising application of 4.5 CV to 5.5 CV (e.g.,about 5 CV) of an equilibration buffer comprising 50 mM to 150 mM (e.g.,about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to9.5 (e.g., about 8.9) to the AEX stationary phase in the column;optionally wherein at least one of steps i)-viii) is performed at alinear velocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr, about300 cm/hr), and/or a residence time of 1.5 min/CV to 4.5 min/CV (e.g.,about 2 min/CV, about 4 min/CV); optionally wherein the rAAV vector is arAAV9 or rAAV3B vector; and optionally wherein the AEX stationary phaseis POROS™ 50 HQ. In some embodiments, at least one of steps i)-vii) isperformed at a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8L/min) through a 6 L to 6.6 L column (e.g., about 6.4 L), or about 314mL/min through a 1.3 L column. One of ordinary skill will understandthat the order of the above steps may be varied.

Load Chase

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) comprises application of a loadchase solution to a column stationary phase following application of thesolution comprising the rAAV vector. A load chase serves to completeapplication of the load or load solution and to remove unbound materialfrom the column. In some embodiments, a load chase serves to removeunbound material from the column. In some embodiments, a load chasesolution comprises 5 mM to 50 mM (e.g., about 20 mM) Tris, pH 8.5 to 9.5(e.g., about 9). In some embodiments, 9 to 11 CV (e.g., about 10 CV) ofa load chase solution is applied to the column stationary phase. In someembodiments, a load chase solution is applied to the column stationaryphase at velocity of 200 cm/hr to 2000 cm/hr (e.g., about 1800 cm/hr)and/or with a residence time of 0.5 minutes/CV. In some embodiments, 9CV to 11 CV (e.g., about 10 CV) of a load chase solution comprising 20mM Tris, pH 9 is applied to AEX stationary phase in a column, optionallyat a velocity of 200 cm/hr to 2000 cm/hr (e.g., about 1800 cm/hr) and/orwith a residence time of about 0.5 minutes/CV.

Gradient Elution

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) comprises recovery of full,intermediate and/or empty capsids by gradient elution. Gradient elutionmay comprise use of at least 2 different solutions (e.g., gradientelution buffers) with different pH, conductivity, and/or modifierconcentration. Over the course of a gradient elution, a percentage of afirst solution is varied in a manner inversely proportional to variationof a percentage of a second solution such that a gradient in the pH,conductivity, and/or modifier concentration is created as the solutionsare mixed and flow through the column stationary phase. For example, atthe start of a gradient elution, a percentage of a first solution (e.g.,a first gradient elution buffer, buffer A) is 100% and a percentage of asecond solution (e.g., a second gradient elution, buffer B) is 0% and atthe end of the gradient elution the percentage of the first solution is0% and the percentage of the second solution is 100%. In anotherembodiment, at the start of a gradient elution, a percentage of a firstsolution (e.g., a first gradient elution buffer, buffer A) is 100% and apercentage of a second solution (e.g., a second gradient elution, bufferB) is 0% and at the end of the gradient elution the percentage of thefirst solution is 25% and the percentage of the second solution is 75%.One of ordinary skill would understand that the percentage of eachsolution at the start of the gradient and at the end of the gradient canbe anywhere between 0% and 100%. For instance, in some embodiments, atthe start of the elution, at the end of the elution, or at any pointover the course of the elution, a percentage of a first gradient elutionbuffer relative to a second gradient elution buffer is about 100%/0%,about 99%/1%, about 98%/2%, about 97%3%, about 96%/4%, about 95%/5%,about 90%10%, about 80%20%, about 75%/25%, about 70%/30%, about 60%/40%,about 50%/50%, about 40%/60%, about 30%/70%, about 25%/75%, about20%/80%, about 10%/90%, about 5%/95%, about 4%/96%, about 3%/97%, about2%/98%, about 1%/99% or about 0%/100%.

In some embodiments, at the start of the elution, at the end of theelution, or at any point over the course of the elution, a percentage ofa first gradient elution buffer relative to a percentage of a secondgradient elution buffer is about 100% to 90%/0% to 10%, 90% to 80%/10%to 20%, 80% to 70%/20% to 30%, 70% to 60%/30% to 40%, 60% to 50%/40% to50%, 50% to 40%/50% to 60%, 40% to 30%/60% to 70%, 30% to 20%/70% to80%, 20% to 10%/80% to 90%, 10% to 0%/90% to 100%.

In some embodiments, over the course of application of 10 to 60 CV of asolution to the column, a percentage of buffer A (e.g., a first gradientelution buffer) is decreased, and a percentage of buffer B (e.g., asecond gradient elution buffer) is increased such that at the end of thegradient elution, the percentage of gradient elution buffer A is 0%, andthe percentage of gradient elution buffer B is 100%. In someembodiments, over the course of application of about 20 CV of a solutionto the column, the percentage of buffer A (e.g., a first gradientelution buffer) is decreased, and the percentage of buffer B (e.g., asecond gradient elution buffer) is increased such that the rate ofincrease of Buffer B is about 5% of buffer B per CV and such that thefinal percentage of buffer B in the solution is 100%. In someembodiments, over the course of application of about 37.5 CV of asolution to the column, the percentage of buffer A (e.g., a firstgradient elution buffer) is decreased, and the percentage of buffer B(e.g., a second gradient elution buffer) is increased such that the rateof increase of Buffer B is about 2% of buffer B per CV, and such thatthe final percentage of buffer B in the solution is 75%.

In some embodiments, over the course of application of 10 to 60 CV of asolution to the column, the percentage of buffer A (e.g., a firstelution buffer) is increased, and the percentage of buffer B (e.g., asecond elution buffer) is decreased such that at the end of the gradientelution, the percentage of gradient elution buffer A is 100%, and thepercentage of gradient elution buffer B is 0%. One of skill in the artwith recognize that a gradient elution may be run to differentpercentages of buffer (e.g., from 0% to 75% buffer B, corresponding to100% to 25% buffer A; from 0% to 50% buffer B, corresponding to 100% to50% buffer A).

In some embodiments, a method of purifying a rAAV vector by AEX of thedisclosure comprises performing gradient elution of a material from astationary phase in a column wherein a concentration of a component of afirst gradient elution buffer or a second gradient elution bufferincreases or decreases continuously during the gradient elution. In someembodiments, a material eluted from the stationary phase comprises arAAV vector to be purified. A rate of increase or decrease of aconcentration of a component of a first gradient elution buffer or asecond gradient elution buffer may be equivalent to a change inconcentration of the component per total CV. In some embodiments, a rateof increase of a concentration of sodium acetate during a gradientelution is equivalent to a change in concentration of the sodium acetateper total CV applied to a stationary phase during the elution. In someembodiments, a change in concentration of a component is relative to aconcentration of the component at the start of a elution as compared toa concentration of the component at the end of the elution. For example,a concentration of a component (e.g., a salt such as sodium acetate) atthe start of a gradient elution is 0 mM to 100 mM, and the concentrationof the component at the end of the elution is 100 mM to 1 M. In someembodiments, a concentration of a salt (e.g., sodium acetate) at thestart of a gradient elution is 0 mM and the concentration of the salt atthe end of the gradient elution is 400 mM to 600 mM (e.g., about 500mM). In some embodiments, a change in a concentration of a component is2 mM to 1 M from the start of a gradient to the end of a gradientelution, over the course of 2 CV to 100 CV of elution buffer. In someembodiments, a change in concentration of a salt is from about 0 mM toabout 500 mM from the start a gradient to the end of a gradient elutionover the course of 10 CV to 60 CV, 10 CV to 50 CV, 10 CV to 40 CV, 10 CVto 30 CV or 15 CV to 25 CV (e.g., 20 CV) of elution buffer, such thatwhen the elution gradient comprises 20 CV of solution, the rate ofchange of sodium acetate concentration is about 500 mM per 20 CV, or 25mM/CV. In some embodiments, a change in concentration of a salt is fromabout 0 mM to about 375 mM from the start a gradient to the end of agradient elution over the course of 10 CV to 60 CV, 10 CV to 50 CV, 10CV to 40 CV, 10 CV to 30 CV or 15 CV to 25 CV (e.g., 37.5 CV) of elutionbuffer, such that when the elution gradient comprises 37.5 CV ofsolution, the rate of change of concentration of sodium acetate is about375 mM per 37.5 CV, or 10 mM/CV.

In some embodiments, during a gradient elution, a concentration ofsodium acetate of a first gradient elution buffer, a second gradientelution buffer or a mixture of both increases continuously during thegradient elution; wherein a rate of increase of the sodium acetate isequivalent to a change in concentration of the sodium acetate per totalCV applied to the stationary phase; and wherein the rate of change inconcentration of the sodium acetate over the gradient elution is about 5mM/CV to 15 mM/CV, 10 mM/CV to 50 mM/CV, 10 mM/CV to 40 mM/CV, 10 mM to30 mM/CV or 20 mM/CV to 30 mM/CV (e.g., about 10 mM/CV, about 25 mM/CV).

In some embodiments, a change in concentration of a component over agradient elution is about 1 mM/CV to 1 M/CV, e.g., 1 mM/CV to 10 mM/CV,1 mM/CV to 25 mM/CV, 5 mM/CV to 15 mM/CV, 10 mM/CV to 50 mM/CV, 50 mM/CVto 100 mM/CV, 100 mM/CV to 500 mM/CV, 500 mM/CV to 1 M/CV, 1 mM/CV to750 mM/CV, 1 mM/CV to 500 mM/CV, 1 mM/CV to 100 mM/CV, 10 mM/CV to 750mM/CV or 50 mM/CV to 500 mM/CV.

In some embodiments, over the course of a gradient elution, aconcentration of a salt in the gradient solution may vary. In someembodiments, over the course of a gradient elution, a concentration of asalt (e.g., sodium chloride, sodium acetate, ammonium acetate, magnesiumchloride, sodium sulfate and a combination thereof) in the gradientsolution may increase or decrease. For example, at the start of agradient elution, a concentration of a salt in the gradient solution maybe 0 mM to 100 mM, and increase to 50 mM to 1 M, e.g., 50 mM to 100 mM,100 mM to 150 mM, 150 mM to 200 mM, 200 mM to 250 mM, 250 mM to 300 mM,300 mM to 400 mM, 400 mM to 500 mM, 500 mM to 600 mM, 600 mM to 700 mM,700 mM to 800 mM, 800 mM to 900 mM, 900 mM to 1 M, 50 mM to 750 mM, 50mM to 500 mM, 50 mM to 400 mM, 50 mM to 200 mM, 100 mM to 1 M, 100 mM to750 mM, 100 mM to 500 mM, 100 mM to 400 mM or 100 mM to 200 mM over thecourse of the gradient elution. In a further example, at the start of agradient elution, a concentration of salt in the gradient solution maybe 50 mM to 1 M, e.g., 50 mM to 100 mM, 100 mM to 150 mM, 150 mM to 200mM, 200 mM to 250 mM, 250 mM to 300 mM, 300 mM to 400 mM, 400 mM to 500mM, 500 mM to 600 mM, 600 mM to 700 mM, 700 mM to 800 mM, 800 mM to 900mM, 900 mM to 1 M, 50 mM to 750 mM, 50 mM to 500 mM, 50 mM to 400 mM, 50mM to 200 mM, 100 mM to 1 M, 100 mM to 750 mM, 100 mM to 500 mM, 100 mMto 400 mM or 100 mM to 200 mM and decrease to 0 mM to 100 mM over thecourse of the gradient elution. In some embodiments, at the start of agradient elution a concentration of sodium acetate in the gradientsolution is about 0 mM and, at the end of the gradient elution theconcentration of sodium acetate is about 500 mM. In some embodiments, atthe start of a gradient elution a concentration of sodium acetate in thegradient elution solution is about 0 mM and, at the end of the gradientelution the concentration of sodium acetate in the gradient elutionsolution is about 375 mM.

In some embodiments, over the course of a gradient elution, a pH of thegradient solution may vary. In some embodiments, over the course of agradient elution, a pH of the gradient solution may increase or maydecrease. In some embodiments, at the start of a gradient elution, a pHof the gradient solution may be between 7.0 and 11.0 (e.g., 7.0 to 7.5,7.5 to 8.0, 8.0 to 8.5, 8.5 to 9.0, 9.0 to 9.5, 10.0 to 10.5, 10.5 to11, 7.5 to 10.5, 8.0 to 10.0, 8.5 to 9.5 or 8.0 to 9.0). In someembodiments, at the end of a gradient elution, a pH of the gradientsolution may be between 7.0 and 11.0 (e.g., 7.0 to 7.5, 7.5 to 8.0, 8.0to 8.5, 8.5 to 9.0, 9.0 to 9.5, 10.0 to 10.5, 10.5 to 11, 7.5 to 10.5,8.0 to 10.0, 8.5 to 9.5 or 8.0 to 9.0).

In some embodiments, over the course of a gradient elution, aconductivity of the gradient solution may vary. In some embodiments,over the course of a gradient elution, a conductivity of the gradientsolution may increase or may decrease. In some embodiments, at the startof a gradient elution, a conductivity of the gradient solution may bebetween 1.0 mS/cm and 2.5 mS/cm, e.g., 1.2 mS/cm and 2.0 mS/cm. In someembodiments, at the end of a gradient elution, a conductivity of thegradient solution may be between 20 mS/cm and 35 mS/cm, e.g., 27 mS/cmand 33 mS/cm. In some embodiments, at the start of a gradient elution aconductivity of the gradient solution is about 1.6 mS/cm and at the endof the gradient elution the conductivity of the gradient solution isabout 30 mS/cm.

In some embodiments, over the course of a gradient elution, aconcentration of a buffer in the gradient solution may vary. In someembodiments, over the course of a gradient elution, a concentration of abuffer (e.g., Tris (e.g., a mixture of Tris Base and Tris-HCl), BIS-Trispropane, diethanolamine, diethylamine, tricine, triethanolamine and/orbicine of) in the gradient solution may increase or decrease. Forexample, at the start of a gradient elution a concentration of a bufferin the gradient solution may range from 10 mM to 500 mM, e.g., from 10mM to 400 mM, from 10 mM to 300 mM, from 10 mM to 200 mM, from 10 mM to50 mM, from 50 mM to 100 mM, from 50 mM to 150 mM, from 100 mM to 200mM, from 100 mM to 400 mM, from 200 mM to 300 mM, from 300 mM to 400 mM,from 400 mM to 500 mM, or more. At the end of a gradient elution aconcentration of a buffer in the gradient solution may range from 10 mMto 500 mM, e.g., from 10 mM to 400 mM, from 10 mM to 300 mM, from 10 mMto 200 mM, from 10 mM to 50 mM, from 50 mM to 100 mM, from 50 mM to 150mM, from 100 mM to 200 mM, from 100 mM to 400 mM, from 200 mM to 300 mM,from 300 mM to 400 mM, from 400 mM to 500 mM, or more.

In some embodiment, over the course of a gradient elution, aconcentration of a detergent in the gradient solution may vary. In someembodiments, over the course of a gradient elution, a concentration of adetergent (e.g., poloxamer 188 (P188), Triton X-100, polysorbate 80(PS80), Brij-35, nonyl phenoxypolyethoxylethanol (NP-40) and acombination thereof) in the gradient solution, may increase or decrease.For example, at the start of a gradient elution a concentration of adetergent (e.g., P188) in the gradient solution may range from 0.005% to1.0%, e.g., from 0.005% to 0.01%, from 0.005% to 0.5%, from 0.01% to1.0%, from 0.01% to 0.5%, from 0.01% to 0.02%, from 0.02% to 0.03%, from0.03% to 0.04%, from 0.04% to 0.05%, from 0.05% to 0.06%, from 0.05% to1.0%, from 0.05% to 0.5%, from 0.07% to 0.08%, from 0.08% to 0.09%, from0.09% to 0.1%, from 0.1% to 0.5%, from 0.1% to 1.0%, from 0.5% to 1.0%.

In some embodiments, at the end of a gradient elution, a concentrationof a detergent (e.g., P188) in the gradient solution may range from0.005% to 1.0%, e.g., from 0.005% to 0.01%, from 0.005% to 0.5%, from0.01% to 1.0%, from 0.01% to 0.5%, from 0.01% to 0.02%, from 0.02% to0.03%, from 0.03% to 0.04%, from 0.04% to 0.05%, from 0.05% to 0.06%,from 0.05% to 1.0%, from 0.05% to 0.5%, from 0.07% to 0.08%, from 0.08%to 0.09%, from 0.09% to 0.1%, from 0.1% to 0.5 from 0.1% to 1.0%, from0.5% to 1.0%.

Over the course of a gradient elution, while one or more aspects of thegradient solution (e.g., a salt concentration) may be varied, otheraspects of the gradient, such as conductivity, pH, buffer concentration,detergent concentration etc., may remain constant. For example, pH of agradient solution may range from 7.0 to 11.0, e.g., from 7.5 to 10.5,from 8.0 to 10.0, from 8.5 to 9.5 or from 8.0 to 9.0, from 7.0 to 7.5,from 7.5 to 8.0, from 8.0 to 8.5, from 8.5 to 9.0, from 9.0 to 9.5, from9.5 to 10, from 10.0 to 10.5 or from 10.5 to 11.0, but be constantthroughout the gradient elution (e.g., a pH of about 8.8, about 8.9,about 9). In some embodiments, a pH of a gradient elution solution isabout 8.9.

In some embodiments, a concentration of a buffer, such as Tris, BIS-Trispropane, bicine and a combination thereof, in a gradient elution mayrange from 10 mM to 500 mM, e.g., from 10 mM to 30 mM, from 10 mM to 50mM, from 50 mM to 100 mM, from 100 mM to 200 mM, from 200 mM to 300 mM,from 300 mM to 400 mM, from 400 mM to 500 mM, from 10 mM to 400 mM, from10 mM to 300 mM, about 10 mM to 200 mM, about 50 mM to about 150 mM ormore, but be constant throughout the gradient elution (e.g., about 20mM, about 100 mM). In some embodiments, a concentration of a buffer,such as Tris, in a gradient elution is 50 mM to 150 mM. In someembodiments, a concentration of a buffer, such as Tris, in a gradientelution solution is about 100 mM.

In some embodiments, a concentration of a detergent, such as poloxamer188 (P188), Triton X-100, polysorbate 80 (PS80), Brij-35, nonylphenoxypolyethoxylethanol (NP-40) and a combination thereof, in agradient elution may range from 0.005% to 0.01%, from 0.005% to 0.5%,from 0.01% to 1.0%, from 0.01% to 0.5%, from 0.01% to 0.02%, from 0.02%to 0.03%, from 0.03% to 0.04%, from 0.04% to 0.05%, from 0.05% to 0.06%,from 0.05% to 1.0%, from 0.05% to 0.5% from 0.07% to 0.08%, from 0.08%to 0.09%, from 0.09% to 0.1%, from 0.1% to 0.5%, from 0.1% to 1.0%, from0.5% to 1.0% but be constant throughout the gradient elution. In someembodiments, a concentration of P188 during a gradient elution is 0.05%to 0.1%. In some embodiments, a concentration of P188 during a gradientelution is about 0.01%.

During a gradient elution, as conditions within the column change, forexample, pH, conductivity, salt concentration and/or modifierconcentration, substances loaded onto the column elute from the columnat varying points during the gradient.

In some embodiments, AAV capsids (e.g., full, intermediate, empty) arebound to a stationary phase during loading a solution comprising thecapsids to be purified. During a gradient elution, as a percentage of agradient elution buffer increases, such that the concentration of a saltincreases (e.g., sodium acetate), full rAAV vectors are preferentiallyreleased (eluted) from the stationary phase, and empty capsids arepreferentially retained on the stationary phase. Empty capsids arereleased in greater amounts as the percentage of the gradient elutionbuffer further increases (along with the salt concentration). Emptycapsids may also be recovered in an AEX column flow-through that is, theunbound fraction. In some embodiments, full and/or intermediate capsidsare recovered in a first elution peak and in a portion of a secondelution peak (e.g., the first ⅔s of a second elution peak) from an AEXcolumn. Elution of full rAAV vector from the stationary phase can bemonitored during a gradient elution by measuring an A260 and A280 of theeluate, such that an increase in the A260/A280 ratio is indicative of anincrease in the presence of full rAAV vector in the eluate.

In some embodiments, performing a gradient elution comprises applicationof about 20 CV of a solution to a column, wherein the solution is bufferA, buffer B or a mixture of buffer A and buffer B and wherein at thestart of the gradient elution, the solution is 100% buffer A and at theend of the step the solution is 100% B, such that a gradient betweenbuffer A and buffer B is created over the course of the elution phase,optionally, wherein the rate of increase of buffer B is about 5% ofbuffer B per CV, and optionally, when buffer B comprises sodium acetate,the concentration of sodium acetate increases at a rate of 25 mM per CV.

In some embodiments, performing a gradient elution comprises applicationof about 37.5 CV of a solution to a column, wherein the solution isbuffer A, buffer B or a mixture of buffer A and buffer B and wherein atthe start of the gradient elution, the solution is 100% buffer A and atthe end of the step the solution is 75% buffer B and 25% buffer A, suchthat a gradient between buffer A and buffer B is created over the courseof the elution phase, optionally, wherein the rate of increase of bufferB is about 2% of buffer B per CV, and optionally, when buffer Bcomprises sodium acetate, the concentration of sodium acetate increaseat a rate of 10 mM per CV.

In some embodiments, buffer A (e.g., a first gradient elution buffer)comprises 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015%(e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9). In someembodiments, buffer B (e.g., a second gradient elution buffer) comprisesabout 400 mM to 600 mM (e.g., about 500 mM) sodium acetate, 50 mM to 150mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188,pH 8.5 to 9.5 (e.g., about 8.9).

In some embodiments, a gradient elution begins with application of 100%buffer A to the column and ends with application of 100% buffer B to thecolumn over the course of 20 CV to 24 CV (e.g., about 20 CV), such thata gradient between buffer A and buffer B is created over the course ofthe elution phase, and wherein buffer A comprises about 100 mM Tris,0.01% P188, pH 8.9 and buffer B comprises about 500 mM sodium acetate,100 mM Tris, 0.01% P188, pH 8.9. In some embodiments, a gradient elutionbegins with application of 100% buffer A to the column and ends withapplication of 75% buffer B and 25% buffer A to the column over thecourse of 30 CV to 40 CV (e.g., about 37.5 CV), such that a gradientbetween buffer A and buffer B is created over the course of the elutionphase, and wherein buffer A comprises about 100 mM Tris, 0.01% P188, pH8.9 and buffer B comprises about 500 mM sodium acetate, 100 mM Tris,0.01% P188, pH 8.9.

In some embodiments, a gradient elution buffer comprises 5 mM to 40 mM(e.g., about 20 mM) Tris, pH 9.0. In some embodiments, a gradientelution buffer comprises 5 mM to 40 mM (e.g., about 20 mM) Tris, 400 mMto 600 mM (e.g., about 500 mM) salt (e.g., NaCl, NaAcetate, NH₄Acetateand Na₂SO₄), pH 9.0. In some embodiments, a gradient elution buffercomprises about 20 mM Tris, 500 mM sodium acetate, pH 9.0.

In some embodiments, a residence time of a gradient elution buffer(e.g., buffer, A, buffer B or a mixture of buffer A and buffer B) in anAEX column is 0.1 min/CV to 15 min/CV, e.g., 0.1 min/CV to 1 min/CV, 1min/CV to 2 min/CV, 1.5 min/CV to 2.5 min/CV, 2 min/CV to 4 min/CV, 4min/CV to 6 min/CV, 6 min/CV to 8 min/CV, or 8 min/CV to 10 min/CV, 10min/CV to 12 min/CV, 12 min/CV to 15 min/CV. In some embodiments, aresidence time of a solution in a column is 0.1 min/CV, about 0.5min/CV, about 1.5 min/CV, about 2.0 min/CV, about 2.5 min/CV, about 3min/CV, about 3.6 min/CV or about 4 min/CV, about 5 min/CV, about 6min/CV, about 7 min/CV, about 8 min/CV, about 9 min/CV or about 10min/CV. In some embodiments, a residence time of a gradient elutionbuffer (e.g., buffer A, buffer B or a mixture of buffer A and buffer B)in a column is about 3.6 min/CV or 4 min/CV. In some embodiments, aresidence time of a gradient elution buffer (e.g., buffer A, buffer B ora mixture of buffer A and buffer B) in a column is about 2.0 min/CV.

In some embodiments, a residence time of a gradient elution buffer(e.g., buffer A, buffer B or a mixture of buffer A and buffer B) in acolumn is 1.5 to 2.5 min/CV (e.g., about 2 min/CV). In some embodiments,a residence time of a gradient elution buffer (e.g., a buffer A, bufferB, or mixture of buffer A and buffer B) in a column is 3.5 to 4.5 min/CV(e.g., about 4 min/CV). In some embodiments, a residence time of agradient elution buffer (e.g., buffer A, buffer B or a mixture of bufferA and buffer B) in a column is about 11 min/CV.

In some embodiments, a linear velocity of a gradient elution buffer(e.g., buffer A, buffer B or a mixture of buffer A and buffer B) througha stationary phase in a column is 50 to 1800 cm/hr, e.g., 50 cm/hr to100 cm/hr, 100 cm/hr to 200 cm/hr, 200 cm/hr to 400 cm/hr, 400 cm/hr to600 cm/hr, 600 cm/hr to 800 cm/hr, 800 cm/hr to 1000 cm/hr, 1000 cm/hrto 1500 cm/hr, or 1500 cm/hr to 1800 cm/hr. In some embodiments, alinear velocity of a gradient elution buffer (e.g., buffer A, buffer Bor a mixture of buffer A and buffer B) through a stationary phase in acolumn is about 298 cm/hr or about 300 cm/hr. In some embodiments, alinear velocity of a gradient elution buffer (e.g., buffer A, buffer Bor a mixture of buffer A and buffer B) through a stationary phase in acolumn is about 75 cm/hr, about 204 cm/hr, about 298 cm/hr, about 300cm/hr, about 597 cm/hr, or about 600 cm/hr. In some embodiments, alinear velocity of a gradient elution buffer (e.g., a buffer A, buffer Bor mixture of buffer A and buffer B) through an AEX stationary phase ina 6.0 L to 6.6 L (e.g., 6.4 L) column is about 270 cm/hr to 330 cm/hr(e.g., about 300 cm/hr).

In some embodiments, a flow rate of a gradient elution buffer (e.g.,buffer A, buffer B or a mixture of buffer A and buffer B) through astationary phase in a column is about 0.2 mL/min to 2.0 L/min e.g., 0.2mL/min to 1 mL/min, 1.0 mL/min to 10 mL/min, 10 mL/min to 100 mL/min,100 mL/min to 500 mL/min, 500 mL/min to 1 L/min, 1 L/min to 1.5 L/min,or 1 L/min to 2 L/min. In some embodiments, a flow rate of a gradientelution buffer (e.g., buffer A, buffer B or a mixture of buffer A andbuffer B) through a stationary phase in a column is about 0.47 mL/min.In some embodiments, a flow rate of a gradient elution buffer (e.g.,buffer A, buffer B or a mixture of buffer A and buffer B) through astationary phase in a column is about 1.67 mL/min. In some embodiments,a flow rate of a gradient elution buffer (e.g., buffer A, buffer B or amixture of buffer A and buffer B) through a stationary phase in a columnis about 314 mL/min. In some embodiments, a flow rate of a gradientelution buffer (e.g., buffer A, buffer B or a mixture of buffer A andbuffer B) through a stationary phase in a column is about 1.8 L/min. Insome embodiments, a flow rate of a gradient elution buffer (e.g., bufferA, buffer B or a mixture of buffer A and buffer B) through a stationaryphase in a column is 1.5 to 2.0 L/min. In some embodiments, a flow rateof a gradient elution buffer (e.g., buffer A, buffer B or a mixture ofbuffer A and buffer B) through an AEX stationary phase in a 1.3 L columnis about 314 mL/min. In some embodiments, a flow rate of a gradientelution buffer (e.g., buffer A, buffer B or a mixture of buffer A andbuffer B) through an AEX stationary phase in a 6.0 L to 6.6 L (e.g., 6.4L) column is about 1.8 L/min.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from a solution (e.g., an affinity eluate) comprisesapplication of a gradient elution buffer to a column comprising POROS™50 HQ stationary phase. In some embodiments, a method of purifying arAAV vector (e.g., AAV9, AAV3B or others) from an affinity eluatecomprises performing a gradient elution beginning with application of100% buffer A (e.g., comprises 50 mM to 150 mM (e.g., about 100 mM)Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g.,about 8.9)) and ending with application of 75% to 100% buffer B (e.g.,50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9)) over 15 to 40 CV (e.g.,about 20 CV, about 37.5 CV) to a column comprising an AEX stationaryphase, wherein the rate of change of the percentage of buffer B duringthe gradient elution is 2% buffer B/CV to 5% buffer B/CV. In someembodiments, the column is a 6.0 L to 6.6 L (e.g., 6.4 L) column.

In some embodiments, a method of purifying a rAAV vector (e.g., AAV9,AAV3B or others) from an affinity eluate comprises performing a gradientelution beginning with application of 100% of a first buffer comprisingabout 100 mM Tris, 0.1% P188, pH 8.9 and ending with application of 75%to 100% of a second buffer comprising 500 mM sodium acetate, 100 mMTris, 0.01% P188, pH 8.9 over 15 CV to 40 CV (e.g., about 20 CV, about37.5 CV) to a column comprising an AEX stationary phase at a linearvelocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr, about 300cm/hr), a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min)and/or a residence time of 1.5 min/CV to 4.5 min/CV (e.g., 2 min/CV, 4min/CV), such that a gradient between the first buffer and the secondbuffer is created over the course of the elution, and wherein the rateof change of the percentage of buffer B during the gradient elution is2% buffer B/CV to 5% buffer B/CV. In some embodiments, the column is a6.0 L to 6.6 L (e.g., 6.4 L) column.

In some embodiments, the present disclosure provides a method ofpurifying a rAAV (e.g., rAAV9, rAAV3B or others) vector by AEX, themethod comprising a step of: i) pre-use flushing comprising applicationof ≥4.5 CV (e.g., about 5 CV) of water for injection to an AEXstationary phase in a column; ii) sanitizing comprising application of 5CV to 10 CV (e.g., about 8 CV) or 14.4 to 17.6 CV (e.g., about 16 CV) ofa solution comprising 0.1 M to 1.0 M (e.g., about 0.5 M) NaOH to the AEXstationary phase in the column, optionally by upward flow; iii)regenerating comprising application of 4.5 to 5.5 CV (e.g., about 5 CV)of a solution comprising 1 M to 3 M (e.g., about 2 M) NaCl, 50 mM to 150mM (e.g., about 100) mM Tris, pH 8.5 to 95. (e.g., about 9) to the AEXstationary phase in the column; iv) equilibration comprising applicationof 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising 50 mM to150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to theAEX stationary phase in the column; v) equilibration comprisingapplication of 4.5 to 5.5 CV (e.g., about 5 CV) of an equilibrationbuffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 400 mM to600 mM (e.g., about 500 mM) sodium acetate, 0.005% to 0.015% (e.g.,about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) to the AEX stationaryphase in the column; vi) equilibration comprising application of ≥4.5 CV(e.g., about 5 CV) of an equilibration buffer comprising 100 mM to 300mM (e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g., 200 mM) Tris,0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8) tothe AEX stationary phase in the column; vii) loading an affinity eluatecomprising the rAAV vector to be purified to the AEX stationary phase inthe column, optionally wherein the eluate has been a) diluted about 14.4to 15.5 fold (e.g., about 15 fold) with a buffer comprising 100 to 300mM (e.g., about 200 mM) histidine, 100 to 300 mM (e.g., about 200 mM)Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.7 to 9.0, andoptionally b) filtered through an in-line 0.2 μm filter prior toapplication to the AEX stationary phase; viii) equilibration comprisingapplication of 4.5 to 5.5 CV (e.g., about 5 CV) of an equilibrationbuffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) to theAEX stationary phase in the column; and/or ix) performing gradientelution of material from the stationary phase in the column beginningwith application of 100% of a first buffer comprising 50 mM to 150 mM(e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH8.5 to 9.0 (e.g., about 8.9) and ending with application of 100% of asecond buffer comprising 400 mM to 600 mM (e.g., about 500 mM) sodiumacetate, 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015%(e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., pH 8.9) to the stationaryphase over 20 CV to 24 CV (e.g., about 20 CV); optionally wherein atleast one of steps i)-ix) is performed at a linear velocity of 270 cm/hrto 330 cm/hr (e.g., about 298 cm/hr, about 300 cm/hr), and/or aresidence time of 1.5 min/CV to 4.5 min/CV (e.g., about 2 min/CV, about4 min/CV); optionally wherein the rAAV vector is a rAAV9 or a rAAV3Bvector; and optionally wherein the AEX stationary phase is POROS™ 50 HQ.In some embodiments, at least one of steps i)-ix) is performed at a flowrate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min) through a 6 L to6.6 L column (e.g., about 6.4 L), or about 314 mL/min through a 1.3 Lcolumn. In some embodiments, material eluted from the stationary phaseduring gradient elution comprises a rAAV vector to be purified. One ofordinary skill will understand that the order of the above steps may bevaried.

Gradient Hold

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from a solution (e.g., an affinity eluate) comprisesapplying a gradient hold solution to a column comprising an AEXstationary phase (e.g., POROS™ 50 HQ) for an extended volume to ensurecomplete gradient formation, preferably following a gradient elution. Insome embodiments, a gradient hold solution comprises at least onecomponent selected from the group consisting of a salt, a buffer, adetergent, an amino acid and a combination thereof. In some embodiments,a gradient hold solution comprises a salt selected from the groupconsisting of sodium chloride, sodium acetate, ammonium acetate,magnesium chloride, sodium sulfate and a combination thereof. In someembodiments, a gradient hold solution comprises a buffer selected fromthe group consisting of Tris, BIS-Tris propane, bicine and a combinationthereof. In some embodiments, a gradient hold solution comprises adetergent selected from the group consisting of as poloxamer 188 (P188),Triton X-100, polysorbate 80 (PS80), Brij-35, nonylphenoxypolyethoxylethanol (NP-40) and a combination thereof. In someembodiments, a gradient hold solution comprises a salt, a buffer and adetergent. In some embodiments, a gradient hold solution comprises anamino acid selected from the group consisting of the amino acid isselected from the group consisting of histidine, arginine, glycine,citrulline and a combination thereof. In some embodiments a gradienthold solution comprises sodium acetate, Tris and P188.

In some embodiments, a gradient hold solution comprises 5 mM to 1 M(e.g., about 500 mM) sodium acetate, 1 mM to 1 M (e.g., about 100 mM)Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g.,about 8.9). In some embodiments, 1 CV to 10 CV, e.g., 1 CV to 3 CV, 1 CVto 5 CV, 4.4 CV to 5.5 CV, 1 CV to 8 CV, or 5 CV to 10 CV of gradienthold solution are applied to a column stationary phase. In someembodiments, 4.5 CV to 5.5 CV (e.g., about 5 CV) of a gradient holdsolution comprising about 500 mM sodium acetate, 100 mM Tris, 0.01%P188, pH 8.9 are applied to an AEX column stationary phase (e.g., POROS™50 HQ) at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 300cm/hr), a flow rate of 0.4 mL/min to 2.0 L/min (e.g., about 1.8 L/min)and/or a residence time of 3.5 to 11 min/CV.

Step Elution

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) comprises a step elution (alsoreferred to as an “isocratic elution”). In some embodiments, a stepelution comprises application of at least one step elution solution to acolumn stationary phase, however, more commonly multiple step elutionsolutions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more) are applied to a column stationary phase.

In some embodiments, a step elution solution comprises at least onecomponent selected from the group consisting of a salt, a buffer, adetergent, an amino acid and a combination thereof. In some embodiments,a step elution solution comprises a salt selected from the groupconsisting of sodium chloride, sodium acetate, ammonium acetate,magnesium chloride, sodium sulfate and a combination thereof. In someembodiments, a step elution solution comprises a buffer selected fromthe group consisting of Tris, BIS-Tris propane, bicine and a combinationthereof. In some embodiments, a step elution solution comprises an aminoacid selected from the group consisting of the amino acid is selectedfrom the group consisting of histidine, arginine, glycine, citrullineand a combination thereof. In some embodiments, a step solutioncomprises a detergent selected from the group consisting of as poloxamer188 (P188), Triton X-100, polysorbate 80 (PS80), Brij-35, nonylphenoxypolyethoxylethanol (NP-40) and a combination thereof. In someembodiments, a step elution solution comprises a salt, a buffer and adetergent. In some embodiments a step elution solution comprises sodiumacetate and Tris.

In some embodiments, a concentration of a buffer (e.g., Tris) in a stepelution solution is about 1 mM to 500 mM, e.g., 1 mM to 10 mM, 10 mM to50 mM, 50 mM to 100 mM, 100 mM to 200 mM, 200 mM to 300 mM, 300 mM to400 mM, 400 mM to 500 mM. In some embodiments, a concentration of Trisin a step elution solution is about 20 mM.

In some embodiments a concentration of a salt (e.g., sodium acetate) ina step elution solution is about 5 mM to 600 mM, e.g., 5 mM to 50 mM. 50mM to 100 mM, 100 mM to 200 mM, 200 mM to 300 mM, 300 mM to 400 mM, 400mM to 500 mM, 500 mM to 600 mM. In some embodiments, a concentration ofsodium acetate in a step elution solution is about 64 mM, about 75 mM,about 85 mM, about 95 mM, about 100 mM, about 105 mM, about 109 mM,about 110 mM, about 150 mM, about 200 mM, about 300 mM, about 400 mM,about 500 mM or more.

In some embodiments, a step elution solution comprises 10 mM to 50 mM(e.g., about 20 mM) Tris, 5 to 600 mM salt, pH 8.9 to 9.1. In someembodiments, a salt is sodium acetate. In some embodiments, at least onestep elution solution comprises a buffer selected from the groupconsisting of 20 mM Tris, 64 mM sodium acetate, pH 9.0; 20 mM Tris, 75mM sodium acetate, pH 9.0; 20 mM Tris, 85 mM sodium acetate, pH 9.0; 20mM Tris, 95 mM sodium acetate, pH 9.0; 20 mM Tris, 100 mM sodiumacetate, pH 9.0; 20 mM Tris, 105 mM sodium acetate, pH 9.0; 20 mM Tris,109 mM sodium acetate, pH 9.0; and 20 mM Tris, 500 mM sodium acetate, pH9.0.

In some embodiments, 1 CV to 20 CV, e.g., 1 CV to 3 CV, 2 CV to 3 CV, 1CV to 8 CV, 4 CV to 11 CV, 5 CV to 10 CV, 10 CV to 20 CV or 15 CV to 20CV of at least one step elution solution are applied to a columnstationary phase. In some embodiments, about 2.5 CV, about 5 CV, about10 CV or about 20 CV of at least one step elution solution are appliedto a column stationary phase.

In some embodiments, a step elution solution is applied to a columnstationary phase at a linear velocity of 50 cm/hr to 2000 cm/hr (e.g.,about 75 cm/hr, about 150 cm/hr, about 204 cm/hr, about 600 cm/hr, about1800 cm/hr). In some embodiments, a residence time of a step elutionsolution within a column stationary phase is 1 min/CV to 15 min/CV(e.g., about 1.5 min/CV, about 6 min/CV, about 12 min/CV).

In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stepelution solutions are applied to a stationary phase in a column. In someembodiments, 1 CV to 20 CV of at least one step elution solution (e.g.,2, 3, 4, 5, etc.) comprising 20 mM Tris, 5 to 600 mM salt (e.g., sodiumacetate), pH 8.9 to 9.1 (e.g., pH 9.0) are applied to an AEX column(e.g., POROS™ 50 HQ) at a linear velocity of 50 cm/hr to 2000 cm/hr anda residence time of 1 min/CV to 15 min/CV.

In some embodiments, a step elution solution may also be a stripsolution, and preferably applied to a column stationary phase as thefinal step elution step. A final step elution solution (i.e., a stripsolution) may be applied to a column stationary phase to cause therelease of a substance (e.g., a rAAV vector) from the stationary phase.In some embodiments, a final step elution solution may have a high saltconcentration (e.g., >450 mM). In some embodiments, a final step elutionsolution may comprise 20 mM Tris, 500 mM salt (e.g., sodium acetate), pH8.9 to 9.1.

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) comprises application of a stripsolution to a column stationary phase, preferably following applicationof at least one step elution solution. In some embodiments, a stripsolution comprises 20 mM Tris, 500 mM sodium acetate, pH 8.9 to 9.1.

Fraction Collection, Neutralization and Pooling

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) by AEX comprises collecting atleast one fraction of eluate from an AEX column to recover and enrichfor full capsids, optionally during a gradient elution. In someembodiments, full capsids are collected in a first elution peak and in aportion of a second elution peak (e.g., the first ⅔ of the secondelution peak). Empty capsids may be recovered in an AEX columnflow-through, that is, the unbound fraction. Empty capsids may also berecovered in an elution peak, though generally at a lower level ascompared to the recovery in a column flow through. Intermediate capsidsmay be recovered with full capsids or empty capsids.

During an elution (e.g., a gradient elution) of an AEX method ofpurifying a rAAV vector, eluate from an AEX column may be collected indiscrete fractions of a particular volume, and/or with a particularattribute (e.g., absorbance at a particular wavelength). For example, avolume of eluate such as 1 mL to 4 L, e.g., 1 mL to 10 mL, 1 mL to 3 L,1 mL to 2 L, 1 mL to 1 L, 1 mL to 100 mL, 10 mL to 50 mL, 50 mL to 100mL, 100 mL to 250 mL, 250 mL to 500 mL, 500 mL to 1 L, 1 L to 1.5 L, 1.5L. to 2 L, 2 L to 3 L, 3 L to 4 L, or more (e.g., about 1 mL, 5 mL, 10mL, 100 mL, 500 mL, 1 L, 2 L, 3 L, 4 L etc.) or specific CV equivalentssuch as ⅛ of a CV to 10 CV, e.g., ⅛ of a CV to 1 CV, 1 CV to 2 CV, 2 CVto 5 CV, 5 CV to 8 CV, 8 CV to 10 CV or more (e.g., ⅛ of a CV, ¼ of aCV, ⅓ of a CV, ½ of a CV, 1 CV, 2 CV, 3 CV, 4 CV, 5 CV, 6 CV, 7 CV, 8CV, 9 CV or more) of eluate may be collected from an AEX column during achromatography step (e.g., gradient elution). In some embodiments, avolume of eluate ≥⅓ CV may be collected from an AEX column during achromatography step. In some embodiments, a volume of eluate of about ½CV may be collected from an AEX column during a chromatography step. Insome embodiments, collecting at least one fraction of eluate from an AEXcolumn during a chromatography step (e.g., a gradient elution) comprisescollecting the eluate when an absorbance (e.g., absorbance at 260 nmand/or 280 nm) of a column-flow through reaches an absorbance threshold(e.g., ≥0.5 mAU/mm path length, e.g., 10 mAU/mm path length). In someembodiments, collecting at least one fraction of eluate from an AEXcolumn during a chromatography step (e.g., a gradient elution) comprisescollecting the eluate when a gradient elution solution comprises aparticular percentage of an elution buffer, for example when thegradient elution solution comprises about 30% to about 35% (e.g., about32%) to about 50% to about 55% (e.g., about 52%) of the second elutionbuffer (e.g., buffer B). In some embodiments, a second elution buffer(e.g., buffer B) comprises 500 mM sodium acetate, 100 mM Tris, 0.01%P188, pH 8.9.

In some embodiments, an eluate is collected in multiple fractions (e.g.,5 fractions, 10 fractions, 20 fractions or more) of a particular volume(e.g., ⅓ CV, ½ CV). In some embodiments, an eluate is collected as asingle fraction. In some embodiments, an eluate is collected in a singlefraction when the A280 of the eluate is ≥0.5 mAU, and optionallycollected for about 2.3 CV.

In some embodiments, collecting at least one fraction eluate from an AEXcolumn comprises measuring an absorbance at 260 nm (A260) and/orabsorbance at 280 nm (A280) of the eluate collected from the column,optionally during a gradient elution. In some embodiments, measuring anabsorbance (e.g., at A260 or A280) of an AEX eluate is performed in-linewith collecting the at least one fraction eluate. In some embodiments,when an eluate collected from an AEX column during a chromatographyelution(e.g., a gradient elution) has an A280 of 0.5 to 10 mAU/mm pathlength, at least one fraction of eluate is collected. In someembodiments, collecting eluate from an AEX column comprises collectingat least one fraction of eluate with a volume of ≥⅓ of a CV. In someembodiments, collecting at least one fraction of eluate (e.g., a firstfraction of eluate) from an AEX column, optionally during a gradientelution, comprises collecting at least one fraction of eluate when theA280 of the eluate is ≥0.5 mAU/mm path length, and wherein a volume ofthe at least one fraction of eluate is ≥⅓ of a CV.

In some embodiments, one to 25 fractions, e.g., 1 to 5 fractions, 5 to10 fraction, 10 to 15 fractions, 15 to 20 fractions or 20 to 25fractions of eluate are collected from an AEX column, optionally duringa gradient elution. In some embodiments, at least one, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, at least25, or more, fractions of eluate are collected from an AEX column. Insome embodiments, at least 10 fractions of eluate, each with a volume of≥⅓ of a CV, are collected from an AEX column, optionally during agradient elution. In some embodiments, at least 20 fractions of eluate,each with a volume of about ½ of a CV, are collected from an AEX column,optionally during a gradient elution.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from an affinity eluate by AEX comprises collectingthe first of about 10 fractions of eluate from an AEX column, optionallyduring a gradient elution, when the A280 of the eluate is >0.5 mAU/mmpath length, and wherein each fraction has a volume of ≥⅓ of a CV.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV3Bor others) from an affinity eluate by AEX comprises collecting the firstof about 20 fractions of eluate from an AEX column, optionally during agradient elution, when a percentage of a second elution buffer (e.g.,buffer B) of the gradient elution solution is about 30% to about 35%(e.g., about 32%) and continuing the collecting until the percentage ofa second elution buffer (e.g., buffer B) is about 50% to 55% (e.g.,about 52%) of the gradient elution solution, and wherein each fractionhas a volume of about ½ of a CV.

In some embodiments, a method purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from an solution (e.g., an affinity eluate) by AEXcomprises adjusting a pH of at least one fraction of eluate collectedfrom an AEX column, optionally during a gradient elution. In someembodiments, adjusting a pH of at least one fraction of eluate isreferred to as a neutralization step. In some embodiments, a pH of atleast one fraction of eluate collected from an AEX column is pH 8.5 to9.1 prior to pH adjustment. In some embodiments, a pH of at least onefraction of eluate is adjusted to a pH of 6.8 to 7.6 (e.g., about pH7.2). In some embodiments, a pH of at least one fraction of eluate isadjusted to a pH of 7.5 to 7.7 (e.g., about pH 7.6).

In some embodiments, a pH of at least one fraction of eluate collectedfrom an AEX column is adjusted to a pH of 6.8 to 7.6 by addition of 14%to 16% (eluate volume weight) (e.g., 14.3% to 14.7%, 14.3% to 15%, 15%to 16%) of a solution comprising 50 mM to 500 mM, e.g., about 50 mM to100 mM, 50 mM to 400 mM, 50 mM to 300 mM, 50 mM to 200 mM, 100 mM to 200mM, 100 mM to 300 mM, 200 mM to 300 mM, 300 mM to 400 mM, or 400 mM to500 mM sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5). In someembodiments, adjusting a pH of at least one fraction of eluate collectedfrom an AEX column, optionally during a gradient elution, comprisesadjustment of the pH to 6.8 to 7.6 (e.g., about pH 7.2) by addition ofan eluate volume weight of 14% to 16% (e.g., about 15%) eluate volumeweight of a solution comprising about 250 mM sodium citrate, pH 3.5. Insome embodiments, a pH of at least one fraction of eluate collected froman AEX column is adjusted by addition of a solution comprising about 50mM citrate, pH 3.6.

In some embodiments, a pH of at least one fraction of eluate collectedfrom an AEX column is adjusted to a pH of about 7.5 to 7.7 by collectingthe at least one faction into a vessel comprising about 0.01 CV to 0.1CV (e.g., about 0.066 CV) of a solution comprising 50 mM to 500 mM,e.g., about 50 mM to 100 mM, 50 mM to 400 mM, 50 mM to 300 mM, 50 mM to200 mM, 100 mM to 200 mM, 100 mM to 300 mM, 200 mM to 300 mM, 300 mM to400 mM, or 400 mM to 500 mM sodium citrate, pH 3.0 to 4.0 (e.g., about3.5). In some embodiments, adjusting a pH of at least one fraction ofeluate collected from an AEX column, optionally during a gradientelution, comprises adjustment of the pH to 7.5 to 7.7 (e.g., about pH7.6) by collecting the at least one fraction into a vessel comprisingabout 0.01 CV to 0.1 CV (e.g., about 0.066 CV) of a solution comprisingabout 250 mM sodium citrate, pH 3.5.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from a solution (e.g., an affinity eluate) by AEXcomprises measuring an absorbance of at least one fraction of eluatecollected from an AEX column, optionally during a gradient elution. Insome embodiments, an absorbance of at least one fraction of eluate ismeasured using analytical size exclusion chromatography (SEC) in a highperformance liquid chromatography (HPLC) system, and measuring theabsorbance at one or more wavelengths (e.g., 260 nm and/or 280 nm).

In some embodiments, measuring an absorbance of at least one fraction ofeluate collected from an AEX column comprises measuring the absorbanceat 260 nm (A260) and 280 nm (A280), and optionally determining anA260/A280 ratio (when measured by SEC, the measurement may be referredto as SEC A260/A280 or A260/A280 (SEC)). An A260/A280 ratio of at leastone fraction of eluate collected from an AEX column is at least 0.5 to2.0, e.g., at least 0.5 to 0.75, 0.75 to 1.0, 1.0 to 1.25, 1.25 to 1.5,0.5 to 1.5, 1.5 to 2.0 or more. An A260/A280 ratio of at least onefraction of eluate collected from an AEX column is at least 0.5, atleast 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, atleast 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1.0, atleast 1.10, at least 1.11, at least 1.12, at least 1.13, at least 1.14,at least 1.15, at least 1.16, at least 1.17, at least 1.18, at least1.19, at least 1.20, at least 1.21, at least 1.22, at least 1.23, atleast 1.24, at least 1.25, at least 1.26, at least 1.27, at least 1.28,at least 1.29, at least 1.30, at least 1.31, at least 1.32, at least1.33, at least 1.34, at least 1.35, at least 1.36, at least 1.37, atleast, 1.38, at least 1.39, at least 1.40 or greater). In someembodiments, an A260/A280 ratio of at least one fraction of eluatecollected from an AEX column, optionally during a gradient elution, isat least 1.25.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from a solution (e.g., an affinity eluate) by AEXcomprises measuring a % of high molecular mass species (HMMS) of atleast one fraction of eluate collected from an AEX column, optionallyduring a gradient elution. In some embodiments, a % of HMMS is measuredby SEC. In some embodiments, a % HMMS of at least one fraction of eluatecollected during AEX purification of rAAV vectors produced in a 250 LSUB ranges from 0% to 10% (e.g., 0% to 3.2%). In some embodiments, a %HMMS of at least one fraction of eluate collected during AEXpurification of rAAV vectors produced in a 2000 L SUB ranges from 0.5%to 15% (e.g., 1.2% to 8.3%).

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from a solution (e.g., an affinity eluate) by AEXcomprises determining a % purity of at least one fraction of eluatecollected from an AEX column, optionally during a gradient elution. Insome embodiments, a % purity is determined by RP-HPLC. In someembodiments, a % purity of at least one fraction of eluate collectedduring AEX purification of rAAV vectors produced in a 250 L SUB rangesfrom 95% to 100% (e.g., 99.1% to 99.4%). In some embodiments, a % purityof at least one fraction of eluate collected during AEX purification ofrAAV vectors produced in a 2000 L SUB ranges from 75% to 100% (e.g.,79.6% to 98.7%).

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from a solution (e.g., an affinity eluate) by AEXcomprises measuring an amount of host cell DNA (HC-DNA) of at least onefraction of eluate collected from an AEX column, optionally during agradient elution. In some embodiments, an amount of HC-DNA is measuredby qPCR. In some embodiments, an amount of HC-DNA of at least onefraction of eluate collected during AEX purification of rAAV vectorsproduced in a 250 L SUB ranges from 0.1 pg/1×10⁹ VG to 20 pg/1×10⁹ VG(e.g., 1.0 pg/1×10⁹ VG to 5.9 pg/1×10⁹ VG). In some embodiments, anamount of HC-DNA of at least one fraction of eluate collected during AEXpurification of rAAV vectors produced in a 2000 L SUB ranges from 0.1pg/1×10⁹ VG to 50 pg/1×10⁹ VG (e.g., 2.7 pg/1×10⁹ VG to 26.5 pg/1×10⁹VG).

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from a solution (e.g., an affinity eluate) by AEXcomprises measuring an amount of host cell protein (HCP) of at least onefraction of eluate collected from an AEX column, optionally during agradient elution. In some embodiments, an amount of HCP is measured byELISA. In some embodiments, an amount of HCP of at least one fraction ofeluate collected during AEX purification of rAAV vectors produced in a250 L SUB ranges from an amount lower than the level of quantification(LLOQ) to 5.78 pg/1×10⁹ VG. In some embodiments, an amount of HCP of atleast one fraction of eluate collected during AEX purification of rAAVvectors produced in a 2000 L SUB is LLOQ.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from a solution (e.g., an affinity eluate) comprisescombining at least two fractions of eluate collected from an AEX column(e.g., during a gradient elution) to form a pooled eluate (also referredto herein as an “AEX pool”). In some embodiments, at least two fractionsof eluate from an AEX column, each having an A260/A280 ratio (e.g.,measured by SEC) of at least 0.5 to 2.0, e.g., at least 0.5 to 0.75,0.75 to 1.0, 1.0 to 1.25, 1.25 to 1.5, 0.5 to 1.5, 1.5 to 2.0 or more.In some embodiments, at least two fractions of eluate from an AEXcolumn, each having an A260/A280 ratio (e.g., measured by SEC) of atleast 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, atleast 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, atleast 1.0, at least 1.10, at least 1.11, at least 1.12, at least 1.13,at least 1.14, at least 1.15, at least 1.16, at least 1.17, at least1.18, at least 1.19, at least 1.20, at least 1.21, at least 1.22, atleast 1.23, at least 1.24, at least 1.25, at least 1.26, at least 1.27,at least 1.28, at least 1.29, at least 1.30, at least 1.31, at least1.32, at least 1.33, at least 1.34, at least 1.35, at least 1.36, atleast 1.37, at least, 1.38, at least 1.39, at least 1.40 or greater),are combined to form a pooled eluate. In some embodiments, combining atleast two fractions of eluate collected from an AEX column, optionallyduring a gradient elution, comprises combining at least two fractions ofeluate, each having an A260/A280 ratio of ≥0.98 to form a pooled eluate.In some embodiments, combining at least two fractions of eluatecollected from an AEX column, optionally during a gradient elution,comprises combining at least two fractions of eluate, each having anA260/A280 ratio of ≥1.0 to form a pooled eluate. In some embodiments,combining at least two fractions of eluate collected from an AEX column,optionally during a gradient elution, comprises combining at least twofractions of eluate, each having an A260/A280 ratio of ≥1.22 to form apooled eluate. In some embodiments, combining at least two fractions ofeluate collected from an AEX column, optionally during a gradientelution, comprises combining at least two fractions of eluate, eachhaving an A260/A280 ratio of ≥1.24 to form a pooled eluate. In someembodiments, combining at least two fractions of eluate collected froman AEX column, optionally during a gradient elution, comprises combiningat least two fractions of eluate, each having an A260/A280 ratio of≥1.25 to form a pooled eluate.

In some embodiments, combining at least two fractions of eluate to forma pooled eluate comprises pooling 2 to 7, 2 to 10, 2 to 15, 2 to 20 or 2to 50 fractions of eluate collected from an AEX column, optionallyduring a gradient elution. In some embodiments, an A260/A280 ratio of apooled eluate is at least 0.5 to 2.0, e.g., at least 0.5 to 0.75, 0.75to 1.0, 1.0 to 1.25, 1.25 to 1.5, 0.5 to 1.5, 1.5 to 2.0 or more. Insome embodiments, an A260/A280 ratio of a pooled eluate is at least 0.5,at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75,at least 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1.0,at least 1.10, at least 1.11, at least 1.12, at least 1.13, at least1.14, at least 1.15, at least 1.16, at least 1.17, at least 1.18, atleast 1.19, at least 1.20, at least 1.21, at least 1.22, at least 1.23,at least 1.24, at least 1.25, at least 1.26, at least 1.27, at least1.28, at least 1.29, at least 1.30, at least 1.31, at least 1.32, atleast 1.33, at least 1.34, at least 1.35, at least 1.36, at least 1.37,at least, 1.38, at least 1.39, at least 1.40 or greater). In someembodiments, an A260/A280 ratio of a pooled eluate is >0.97. In someembodiments, an A260/A280 ratio of a pooled eluate is 0.97 to 1.03. Insome embodiments, an A260/A280 ratio of a pooled eluate is 1.0 to 1.05.In some embodiments, an A260/A280 ratio of a pooled eluate is 1.20 to1.40. In some embodiments, an A260/A280 ratio of a pooled eluate is≥1.25, for instance about 1.28 to 1.35, and is enriched for full capsidsas compared to the affinity eluate or diluted affinity eluate prior topurification by AEX.

In some embodiments, a pooled eluate comprises only a single fraction,for example, when only a single fraction meets a predeterminedcriterion, such as a A280 value or A260/A280 ratio. In some embodiments,a pooled eluate comprises only a single fraction, for example, when asingle fraction is collected over the course of performing a gradientelution, starting at a particular point (e.g., when a particular A280value is measured) and ending at a particular point (e.g., a particularA280 value is measured, a specific volume of eluate is collected).

In some embodiments, a pooled eluate has a pH of about 6.5 to 8, 6.8 to7.6, about 6.8 to 7.8, 7.0 to 7.6, about 7.0 to 7.4 or about 7.0 to 7.2.In some embodiments a pooled eluate has a pH of about 6.8 to 7.6.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from an affinity eluate comprises i) collecting thefirst of at least one (e.g., about 10) fraction of eluate from an AEXcolumn during a chromatography step (e.g., a gradient elution) when theA280 of the eluate is >0.5 mAU/mm path length, and wherein a volume ofthe at least one fraction of eluate is equivalent to ⅛ of a CV to 2 CV(e.g., about ⅓ of a CV); ii) adjusting the pH of the at least one (e.g.,about 10) fraction of eluate from the column to a pH of 6.8 to 7.6 byaddition of 14.3% to 15% (eluate volume weight) of a solution comprisingabout 200 mM to 300 mM (e.g., about 250 mM) sodium citrate, pH 3.0 to4.0 (e.g., about 3.5); iii) measuring an absorbance of at least onefraction of eluate collected from the column and determining anA260/A280 ratio; and/or iv) combining at least two fractions of eluatecollected from the column to form a pooled eluate, wherein an A260/A280of each of the at least two fractions of eluate is ≥1.25; wherein anA260/A280 of the pooled eluate is ≥1.25, (e.g., about 1.28 to 1.35), andoptionally wherein a pH of the at least one fraction of eluate or thepooled eluate is 6.8 to 7.6, and wherein the at least one fraction ofeluate or the pooled eluate is enriched for full capsids, and/ordepleted of empty capsids, as compared to an affinity eluate or adiluted, and optionally filtered affinity eluate prior to purificationby AEX.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from an affinity eluate comprises i) collecting thefirst of at least one fraction (e.g., about 20) of eluate from an AEXcolumn during a gradient elution step when a gradient elution solutioncomprises about 65% to about 70% (e.g., about 68%) of a first elutionbuffer (e.g., buffer A) comprising about 100 mM Tris, about 0.01% P188,pH 8.9 and about 30% to about 35% (e.g., about 32%) of a second elutionbuffer (e.g., buffer B) comprising about 500 mM sodium acetate, about100 mM Tris, about 0.01% P188, pH 8.9 and continuing to collect allfractions of eluate until the percentage of the first elution buffer isabout 45% to about 50% (e.g., about 48%) and the percentage of thesecond elution buffer is about 50% to about 55% (e.g., about 52%), andwherein a volume of the at least one fraction of eluate is equivalent to⅛ of a CV to 2 CV (e.g., about ½ of a CV); ii) adjusting the pH of theat least one fraction (e.g., about 20) of eluate from the column to a pHof 7.5 to 7.7 by collecting the at least one fraction of eluate into avessel comprising 0.01 CV to 0.1 CV (e.g., about 0.066 CV) of a solutioncomprising about 200 mM to 300 mM (e.g., about 250 mM) sodium citrate,pH 3.0 to 4.0 (e.g., about 3.5); iii) measuring an absorbance of atleast one fraction of eluate collected from the column and determiningan A260/A280 ratio; and/or iv) combining at least two fractions ofeluate collected from the column to form a pooled eluate, wherein anA260/A280 of each of the at least two fractions of eluate is ≥0.98;wherein an A260/A280 of the pooled eluate is ≥1.0, and wherein the atleast one fraction of eluate or the pooled eluate is enriched for fullcapsids, and/or depleted of empty capsids, as compared to an affinityeluate or a diluted affinity eluate prior to purification by AEX.

In some embodiments, the present disclosure provides a method ofpurifying an rAAV (e.g., rAAV9, rAAV3B or others) vector by AEX, themethod comprising a step of: i) pre-use flushing comprising applicationof ≥4.5 CV (e.g., about 5 CV) of water for injection to an AEXstationary phase in a column; ii) sanitizing comprising application of14.4 CV to 17.6 CV (e.g., about 16 CV) of a solution comprising 0.1 M to1.0 M (e.g., about 0.5 M) NaOH to the AEX stationary phase in thecolumn, optionally by upward flow; iii) regenerating comprisingapplication of 4.5 CV to 5.5 CV (e.g., about 5 CV) of a solutioncomprising 1 M to 3 M (e.g., about 2 M) NaCl, 50 mM to 150 mM (e.g.,about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about pH 9) to the AEXstationary phase in the column; iv) equilibration comprising applicationof 4.5 CV to 5.5 CV (e.g., about 5 CV) of a solution comprising 50 mM to150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to theAEX stationary phase in the column; v) equilibration comprisingapplication of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibrationbuffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 400 mM to600 mM (e.g., about 500 mM) sodium acetate, 0.005% to 0.015% (e.g.,about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) to the AEX stationaryphase in the column; vi) equilibration comprising application of ≥4.5 CV(e.g., about 5 CV) of an equilibration buffer comprising 100 mM to 300mM (e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g., about 200 mM)Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about8.8) to the AEX stationary phase in the column; vii) loading an affinityeluate comprising the rAAV vector to be purified to the AEX stationaryphase in the column, optionally wherein the eluate has been a) dilutedabout 14.4 to 15.5 fold (e.g., about 15 fold) with a buffer comprising100 mM to 300 mM (e.g., about 200 mM) histidine, 100 mM to 300 mM.(e.g., about 200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.7to 9.0, and optionally b) filtered through an in-line 0.2 μm filterprior to application to the stationary phase; viii) equilibrationcomprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of anequilibration buffer comprising 50 mM to 150 mM (e.g., about 100 mM)Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g.,about 8.9) to the AEX stationary phase in the column; ix) performinggradient elution of a material from the stationary phase in the columnbeginning with application of 100% of a first buffer comprising 50 mM to150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%)P188, pH 8.5 to 9.5 (e.g., 8.9) and ending with application of 100% of asecond buffer comprising 400 mM to 600 mM (e.g., about 500 mM) sodiumacetate, 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015%(e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., pH 8.9) to the stationaryphase over 20 CV to 24 CV (e.g., about 20 CV); x) collecting the firstof at least one (e.g., about 10) fraction of eluate from the columnduring the gradient elution, when the A280 of the eluate has a ≥0.5mAU/mm path length, and wherein a volume of the at least one fraction ofeluate is equivalent to ⅛ to 2 CV (e.g., about ⅓ of a CV); xi) adjustinga pH of at least one (e.g., about 10) fraction of eluate from the columnto a pH of 6.8 to 7.6, optionally, by addition of 14.3% to 15% (eluatevolume weight) of a solution comprising 200 mM to 300 mM (e.g., about250 mM) sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5); xii) measuringan absorbance of at least one fraction of eluate collected from thecolumn and determining an A260/A280 ratio (e.g., by SEC); and/or xiii)combining at least two fractions of eluate fraction collected from thecolumn to form a pooled eluate, wherein an A260/A280 of the at least onefraction of eluate is ≥1.25, wherein an A260/A280 of the pooled eluateis ≥1.25, (e.g., about 1.28 to 1.35), and optionally wherein a pH of theat least one fraction of eluate or the pooled eluate is 6.8 to 7.6, andwherein the at least one fraction of eluate or the pooled eluate isenriched for full capsids, and/or depleted of empty capsids, as comparedto the affinity eluate or the diluted, and optionally filtered affinityeluate; optionally wherein at least one of steps i) to ix) is performedat a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 300 cm/hr),a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min) through a6.0 L to 6.6 L (e.g., about 6.4 L) column, or about 314 mL/min through a1.3 L column, and/or a residence time of 3.5 min/CV to 4.5 min/CV (e.g.,about 4 min/CV); optionally wherein the rAAV vector is an AAV9 vector;and optionally wherein the AEX stationary phase is POROS™ 50 HQ. In someembodiments, a material eluted from the stationary phase during gradientelution includes a rAAV vector to be purified.

In some embodiments, the present disclosure provides a method ofpurifying an rAAV (e.g., rAAV9, or AAV3B or others) vector by AEX, themethod comprising a step of: i) sanitizing comprising application of 5CV to 10 CV (e.g., about 8 CV) of a solution comprising 0.1 M to 1.0 M(e.g., about 0.5 M) NaOH to the AEX stationary phase in the column; ii)regenerating comprising application of 4.5 CV to 5.5 CV (e.g., about 5CV) of a solution comprising 1 M to 3 M (e.g., about 2 M) NaCl, 50 mM to150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about pH 9) tothe AEX stationary phase in the column; iii) equilibration comprisingapplication of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibrationbuffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 400 mM to600 mM (e.g., about 500 mM) sodium acetate, 0.005% to 0.015% (e.g.,about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) to the AEX stationaryphase in the column; iv) equilibration comprising application of ≥4.5 CV(e.g., about 5 CV) of an equilibration buffer comprising 100 mM to 300mM (e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g., about 200) mMTris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about8.8) to the AEX stationary phase in the column; v) loading an affinityeluate comprising the rAAV vector to be purified to the AEX stationaryphase in the column, optionally wherein the eluate has been dilutedabout 14.4 to 15.5 fold (e.g., about 15 fold) with a buffer comprising100 mM to 300 mM (e.g., about 200 mM) histidine, 100 mM to 300 mM.(e.g., about 200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.7to 9.0; vi) equilibration comprising application of 4.5 CV to 5.5 CV(e.g., about 5 CV) of an equilibration buffer comprising 50 mM to 150 mM(e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH8.5 to 9.5 (e.g., about 8.9) to the AEX stationary phase in the column;vii) performing gradient elution of a material from the stationary phasein the column beginning with application of 100% of a first buffercomprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015%(e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9) and ending withapplication of 75% of a second buffer comprising 400 mM to 600 mM (e.g.,about 500 mM) sodium acetate, 50 mM to 150 mM (e.g., about 100 mM) Tris,0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., pH 8.9)to the stationary phase over 20 to 40 CV (e.g., about 37.5 CV); viii)collecting the first of at least one fraction (e.g., about 20) of eluatefrom the column during the gradient elution, when the percentage of thefirst buffer is about 65% to about 70% (e.g., about 68%) and when thepercentage of the second buffer is about 30% to 35% (e.g., about 32%)and continuing to collect all fractions of eluate until the percentageof the first buffer is about 45% to about 50% (e.g., about 48%) and thepercentage of the second buffer is about 50% to 55% (e.g., about 52%),and wherein a volume of the at least one fraction of eluate isequivalent to ⅛ to 2 CV (e.g., about ½ of a CV); ix) adjusting a pH ofat least one (e.g., about 20) fraction of eluate from the column to a pHof 7.5 to 7.7, optionally, by collecting the at least one fraction ofeluate into a vessel comprising about 0.01 CV to 0.1 CV (e.g., about0.066 CV) of a solution comprising 200 mM to 300 mM (e.g., about 250 mM)sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5); x) measuring anabsorbance of at least one fraction of eluate collected from the columnand determining an A260/A280 ratio (e.g., by SEC); and/or xii) combiningat least two fractions of eluate fraction collected from the column toform a pooled eluate, wherein an A260/A280 of the at least one fractionof eluate is ≥0.98, wherein an A260/A280 of the pooled eluate is ≥1.0,and wherein the at least one fraction of eluate or the pooled eluate isenriched for full capsids, and/or depleted of empty capsids, as comparedto the affinity eluate or the filtered affinity eluate; optionallywherein at least one of steps i) to vii) is performed at a linearvelocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr) and/or aresidence time of 1.5 min/CV to 4.5 min/CV (e.g., about 2 min/CV);optionally wherein the rAAV vector is a rAAV3B vector; and optionallywherein the AEX stationary phase is POROS™ 50 HQ. In some embodiments, amaterial eluted from the stationary phase during gradient elutionincludes a rAAV vector to be purified.

In some embodiments, the present disclosure provides a method ofpurifying an rAAV (e.g., rAAV9 or others) vector by AEX, the methodcomprising a step of: i) pre-use flushing comprising application of ≥4.5CV (e.g., about 5 CV) of water for injection to an AEX stationary phasein a column; ii) sanitizing comprising application of 14.4 CV to 17.6 CV(e.g., about 16 CV) of a solution comprising 0.1 M to 1.0 M (e.g., about0.5 M) NaOH to the AEX stationary phase in the column, optionally byupward flow; iii) regenerating comprising application of 4.5 CV to 5.5CV (e.g., about 5 CV) of a solution comprising 1 M to 3 M (e.g., about 2M) NaCl, 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g.,about pH 9) to the AEX stationary phase in the column; iv) equilibrationcomprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of asolution comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to9.5 (e.g., about 9) to the AEX stationary phase in the column; v)equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5CV) of an equilibration buffer comprising 50 mM to 150 mM (e.g., about100 mM) Tris, 400 mM to 600 mM (e.g., about 500 mM) sodium acetate,0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about8.9) to the AEX stationary phase in the column; vi) equilibrationcomprising application of ≥4.5 CV (e.g., about 5 CV) of an equilibrationbuffer comprising 100 mM to 300 mM (e.g., about 200 mM) histidine, 100mM to 300 mM (e.g., about 200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%)P188, pH 8.5 to 9.5 (e.g., about 8.8) to the AEX stationary phase in thecolumn; vii) loading an affinity eluate comprising the rAAV vector to bepurified to the AEX stationary phase in the column, optionally whereinthe eluate has been a) diluted about 14.4 to 15.5 fold (e.g., about 15fold) with a buffer comprising 100 mM to 300 mM (e.g., about 200 mM)histidine, 100 mM to 300 mM (e.g., about 200 mM) Tris, 0.1% to 1.0%(e.g., about 0.5%) P188, pH 8.7 to 9.0, and optionally b) filteredthrough an in-line 0.2 μm filter prior to application to the stationaryphase; viii) equilibration comprising application of 4.5 CV to 5.5 CV(e.g., about 5 CV) of an equilibration buffer comprising 50 mM to 150 mM(e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH8.5 to 9.5 (e.g., about 8.9) to the AEX stationary phase in the column;ix) performing gradient elution of a material from the stationary phasein the column beginning with application of 100% of a first buffercomprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.15%(e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9) and ending withapplication of 100% of a second buffer comprising 400 mM to 600 mM(e.g., about 500 mM) sodium acetate, 50 mM to 150 mM (e.g., about 100mM) Tris, 0.005% to 0.015% (e.g., about P188, pH 8.5 to 9.5 (e.g., pH8.9) to the stationary phase over 20 CV to 24 CV (e.g., about 20 CV); x)collecting a fraction of eluate from the column during the gradientelution, when the A280 of the eluate is ≥0.5 mAU/mm path length, andwherein a volume of the fraction of eluate is equivalent to ⅛ to 2 CV(e.g., about ⅓ of a CV); and/or xi) adjusting a pH of the fraction ofeluate from the column to a pH of 6.8 to 7.6, optionally, by addition of14.3% to 15% (eluate volume weight) of a solution comprising about 200mM to 300 mM (e.g., about 250 mM) sodium citrate, pH 4.0 to 4.5 (e.g.,about 3.5); and wherein the fraction of eluate is enriched for fullcapsids, and/or depleted of empty capsids, as compared to the affinityeluate or the diluted, and optionally filtered affinity eluate;optionally wherein at least one of steps i) to ix) is performed at alinear velocity of 270 cm/hr to 330 cm/hr (e.g., about 300 cm/hr), aflow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min) through a6.0 L to 6.6 L (e.g., about 6.4 L) column, or about 314 mL/min through a1.3 L column, and/or a residence time of 3.5 min/CV to 4.5 min/CV (e.g.,about 4 min/CV); optionally wherein the rAAV vector is an AAV9 vector;and optionally wherein the AEX stationary phase is POROS™ 50 HQ. In someembodiments, a material eluted from the stationary phase during gradientelution includes a rAAV vector to be purified.

In some embodiments, the present disclosure provides a method ofpurifying an rAAV (e.g., rAAV9, AAV3B or others) vector by AEX, themethod comprising a step of: i) sanitizing comprising application of 5CV to 10 CV (e.g., about 8 CV) of a solution comprising 0.1 M to 1.0 M(e.g., about 0.5 M) NaOH to the AEX stationary phase in the column; ii)regenerating comprising application of 4.5 CV to 5.5 CV (e.g., about 5CV) of a solution comprising 1 M to 3 M (e.g., about 2 M) NaCl, 50 mM to150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about pH 9) tothe AEX stationary phase in the column; iii) equilibration comprisingapplication of 4.5 CV to 5.5 CV (e.g., about 5 CV) of a solutioncomprising 50 mM to 150 mM (e.g., 100 mM) Tris, pH 8.5 to 9.5 (e.g.,about 9) to the AEX stationary phase in the column; iv) equilibrationcomprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of anequilibration buffer comprising 50 mM to 150 mM (e.g., about 100 mM)Tris, 400 mM to 600 mM (e.g., about 500 mM) sodium acetate, 0.005% to0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) to theAEX stationary phase in the column; v) equilibration comprisingapplication of ≥4.5 CV (e.g., about 5 CV) of an equilibration buffercomprising 100 mM to 300 mM (e.g., about 200 mM) histidine, 100 mM to300 mM (e.g., about 200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188,pH 8.5 to 9.5 (e.g., about 8.8) to the AEX stationary phase in thecolumn; vi) loading an affinity eluate comprising the rAAV vector to bepurified to the AEX stationary phase in the column, optionally whereinthe eluate has been diluted about 14.4 to 15.5 fold (e.g., about 15fold) with a buffer comprising 100 mM to 300 mM (e.g., about 200 mM)histidine, 100 mM to 300 mM (e.g., about 200 mM) Tris, 0.1% to 1.0%(e.g., about 0.5%) P188, pH 8.7 to 9.0; vii) equilibration comprisingapplication of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibrationbuffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to0.015% (e.g., about 0.01%) P188, pH 8.4 to 9.5 (e.g., about 8.9) to theAEX stationary phase in the column; viii) performing gradient elution ofa material from the stationary phase in the column beginning withapplication of 100% of a first buffer comprising 50 mM to 150 mM (e.g.,about 100 mM) Tris, 0.005% to 0.15% (e.g., about 0.01%) P188, pH 8.5 to9.5 (e.g., 8.9) and ending with application of 75% of a second buffercomprising 400 mM to 600 mM (e.g., about 500 mM) sodium acetate, 50 mMto 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about0.01%) P188, pH 8.5 to 9.5 (e.g., pH 8.9) to the stationary phase over20 CV to 40 CV (e.g., about 37.5 CV); ix) collecting a fraction ofeluate from the column during the gradient elution when the percentageof the first buffer is about 65% to about 70% (e.g., about 68%) and whenthe percentage of the second buffer is about 30% to about 35% (e.g.,about 32%) and continuing to collect all fractions of eluate until thepercentage of the first buffer is about 45% to about 50% (e.g., about48%) and the percentage of the second buffer is about 50% to 55% (e.g.,about 52%), and wherein a volume of the fraction of eluate is equivalentto ⅛ to 2 CV (e.g., about ½ of a CV); and/or x) adjusting a pH of thefraction of eluate from the column to a pH of 7.5 to 7.7, optionally, bycollecting the fraction of elute into a vessel comprising about 0.01 CVto 0.1 CV (e.g., about 0.066 CV) of a solution comprising 200 mM to 300mM (e.g., about 250 mM) sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5);and wherein the fraction of eluate is enriched for full capsids, and/ordepleted of empty capsids, as compared to the affinity eluate or thediluted affinity eluate; optionally wherein at least one of steps i) toviii) is performed at a linear velocity of 270 cm/hr to 330 cm/hr (e.g.,about 298 cm/hr) and/or a residence time of 1.5 min/CV to 4.5 min/CV(e.g., about 2 min/CV); optionally wherein the rAAV vector is an AAV3Bvector; and optionally wherein the AEX stationary phase is POROS™ 50 HQ.In some embodiments, a material eluted from the stationary phase duringgradient elution includes a rAAV vector to be purified.

Characterization of Pooled Eluate and Drug Substance

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) by AEX comprises collecting atleast one fraction of eluate from an AEX column during an elution step(e.g., a gradient elution) and forming a pooled eluate enriched for fullcapsids as compared to a percentage of full capsids in the solution. Amethod of purifying a rAAV vector from a solution by AEX comprisingcollecting at least one fraction of eluate from the AEX column during anelution step and forming a pooled eluate further comprises filtering thepooled eluate by a method selected from the group consisting of viralfiltration, ultrafiltration/diafiltration (UF/DF), filtration through a0.2 μm filter and a combination thereof, to produce a drug substance. Insome embodiments, quality attributes, including A260/A280 (e.g., asmeasured by SEC), percentages of full capsid, intermediate capsid andempty capsid, % purity, % HMMS, amount of HCP and/or amount of HC-DNA ofa pooled eluate are not substantially different from the same qualityattribute of a drug substance produced from the pooled eluate.

In some embodiments, the percentage of full capsids in an affinityeluate comprising an rAAV vector to be purified is less than 20% oftotal capsids. In some embodiments, a pooled eluate or drug substanceprepared by methods disclosed herein is enriched for full capsids suchthat full capsids comprise 20% to 60%, 20% to 70%, 20% to 80%, 20% to90%, 20% to 95%, 20% to 98%, 20% to 99%, 20% to greater than 99%, 40% to50%, 40% to 60%, 40% to 70%, 40% to 80% (e.g., 44%, 45%, 50%, 53%) oftotal capsids in the pooled eluate or drug substance, and optionallywherein the capsids are measured by analytical ultracentrifugation (AUC)(Burnham B. et al. Human Gene Therapy Methods (2015) 26; 228-242). Insome embodiments, a pooled eluate or drug substance prepared by methodsdisclosed herein is enriched for full capsids such that full capsidscomprise 52+/−7% of total capsids in the pooled eluate or drugsubstance. In some embodiments, a method of purifying a rAAV vector froman affinity eluate comprises increasing the percentage of full capsidsfrom less than 30% (e.g., 12% to 25%) in an affinity eluate to greaterthan 30% (e.g., 40% to 55%, 45% to 65%, 40% to greater than 99%) oftotal capsids in a pooled AEX eluate or drug substance.

In some embodiments, a pooled AEX eluate prepared by methods disclosedherein is enriched for full capsids such that full capsids comprise22.9+/−2.9% of total capsids in the pooled eluate. In some embodiments,a method of purifying a rAAV vector from an affinity eluate comprisesincreasing the percentage of full capsids from less than 20% (e.g., 10%to 19%) in an affinity eluate to 20% or greater (e.g., 20% to 30%, 30%to 40%, 40% to 55%, 45% to 65%, 40% to greater than 99%) of totalcapsids in a pooled AEX eluate. In some embodiments, a method ofpurifying a rAAV vector from an affinity eluate comprises increasing thepercentage of full capsids from 11.1±2.1 in an affinity eluate to22.9±2.9% of total capsids in a pooled AEX eluate.

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) by AEX comprises collecting atleast on fraction of eluate from an AEX column during an elution step(e.g., a gradient elution) and forming a pooled eluate with a depletedpercentage of empty capsids as compared to the percentage of emptycapsids in the solution, and wherein the pooled eluate is furthersubjected to a method of filtration selected from the group consistingof viral filtration, ultrafiltration/diafiltration (UF/DF), filtrationthrough a 0.2 μm filter and a combination thereof, to produce a drugsubstance. In some embodiments, a percentage of empty capsids in anaffinity eluate comprising an rAAV vector to be purified is 70% orgreater of total capsids. In some embodiments, a pooled eluate or drugsubstance prepared by methods disclosed herein is depleted of emptycapsids such that empty capsids comprise 10% to 65%, 10% to 60%, 10% to50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 65%, 20% to 60%, 20% to50%, 20% to 40%, or 18% to 29%, (e.g., ≤29%) of total capsids in thepooled eluate or drug substance, and optionally wherein the capsids aremeasured by analytical ultracentrifugation (AUC). In some embodiments, apooled eluate or drug substance prepared by methods disclosed herein isdepleted of empty capsids such that empty capsids comprise 20%+/−7% oftotal capsids in the pooled eluate or drug substance. In someembodiments, a method of purifying a rAAV vector from an affinity eluatecomprises reducing a percentage of empty capsids from 40% to 90% in anaffinity eluate, to ≤30% of total capsids in a pooled AEX eluate or drugsubstance. In some embodiments, a method of purifying a rAAV vector froman affinity eluate comprises reducing a percentage of empty capsids from79.7±2.5% in an affinity eluate, to 67.5±3.8% of total capsids in apooled AEX eluate or drug substance.

A method of purifying a rAAV vector (e.g., rAAV9, AAV3B or others) froma solution (e.g., an affinity eluate) by AEX comprises collecting atleast one fraction of eluate from an AEX column during an elution step(e.g., a gradient elution) and forming a pooled eluate comprisingintermediate capsids, and wherein the pooled eluate is further subjectedto a method of filtration selected from the group consisting of viralfiltration, ultrafiltration/diafiltration (UF/DF), filtration through a0.2 μm filter and a combination thereof, to produce a drug substance. Insome embodiments, intermediate capsids comprise 10% to 65%, 10% to 60%,10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 65%, 20% to 60%,20% to 50%, 20% to 40%, or 18% to 22% of total capsids in a pooledeluate or drug substance, and optionally wherein the capsids aremeasured by analytical ultracentrifugation (AUC). In some embodiments,intermediate capsids comprise 28%+/−5% of total capsids in a pooledeluate or drug substance. In some embodiments, intermediate capsidscomprise 9.6%+/−1.4% of total capsids in a pooled eluate or drugsubstance.

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) by AEX comprises collecting atleast one fraction of eluate from an AEX column during an elution step(e.g., a gradient elution) and forming a pooled eluate or drug substancethat is enriched for full capsids and depleted of empty capsids ascompared to the percentage of full capsids and empty capsids in thesolution comprising the rAAV vector to be purified. In addition to fullcapsids and empty capsids, capsids which contain a partial vector genome(also referred to as a truncated, or fragmented vector genome) and/ornon-transgene-related DNA (i.e., intermediate capsids) may, in certainnon-limiting exemplary embodiments, make up the balance of capsidspecies in a pooled eluate (e.g., a pooled AEX eluate) or drugsubstance.

In some embodiments, a method of purifying a rAAV vector from anaffinity eluate by AEX produces a pooled eluate or drug substancecomprising about 53% full rAAV capsids, about 23% intermediate capsidsand about 24% empty capsids of total capsids.

In some embodiments, a method of purifying a rAAV vector from anaffinity eluate by AEX produces a pooled eluate or drug substancecomprising about 44% full rAAV capsids, about 27% intermediate capsidsand about 29% empty capsids of total capsids.

In some embodiments, a method of purifying a rAAV vector from anaffinity eluate by AEX produces a pooled eluate or drug substancecomprising 20% to >99% full rAAV capsids, 5% to 65% intermediate capsidsand 10% to 65% empty capsids of total capsids.

In some embodiments, a method of purifying a rAAV vector from anaffinity eluate by AEX produces a pooled eluate or drug substancecomprising 45% to 65% full rAAV capsids, 19% to 28% intermediate capsidsand 10% to 37% empty capsids. In some embodiments, the affinity eluateis generated from affinity chromatography purification of a rAAV vectorproduced in a vessel with a volume of 100 L to 500 L (e.g., about 250L), optionally, wherein the vessel is a SUB.

In some embodiments, a pooled eluate or drug substance prepared bymethods disclosed herein is enriched for full capsids such that fullcapsids comprise 55%+/−7% of total capsids in the pooled eluate or thedrug substance. In some embodiments, the rAAV vector present in thepooled eluate or drug substance is produced in a vessel with a volume of100 L to 500 L (e.g., about 250 L), optionally, wherein the vessel isSUB.

In some embodiments, a pooled eluate or drug substance prepared bymethods disclosed herein comprises 24%+/−3% intermediate capsids oftotal capsids in the pooled eluate or the drug substance. In someembodiments, the rAAV vector present in the pooled eluate or drugsubstance is produced in a vessel with a volume of 100 L to 500 L (e.g.,about 250 L), optionally, wherein the vessel is SUB.

In some embodiments, a pooled eluate or drug substance prepared bymethods disclosed herein is depleted of empty capsids such that emptycapsids comprise 21%+/−10% of total capsids in the pooled eluate or thedrug substance. In some embodiments, the rAAV vector present in thepooled eluate or drug substance is produced in a vessel with a volume of100 L to 500 L (e.g., about 250 L), optionally, wherein the vessel isSUB.

In some embodiments, a method of purifying a rAAV vector from anaffinity eluate by AEX produces a pooled eluate or drug substancecomprising 45% to 52% full rAAV capsids, 27% to 37% intermediate capsidsand/or 18% to 22% empty capsids. In some embodiments, the affinityeluate is generated from affinity chromatography purification of a rAAVvector produced in a vessel of about 1000 L to 3000 L (e.g., about 2000L), optionally, wherein the vessel is a SUB.

In some embodiments, a pooled eluate or drug substance prepared bymethods disclosed herein is enriched for full capsids such that fullcapsids comprise 49%+/−2% of total capsids in the pooled eluate or thedrug substance. In some embodiments, the rAAV vector present in thepooled eluate or drug substance is produced in a vessel with a volume of1000 L to 3000 L (e.g., about 2000 L), optionally, wherein the vessel isSUB.

In some embodiments, a pooled eluate or drug substance prepared bymethods disclosed herein comprises 32%+/−4% intermediate capsids oftotal capsids in the pooled eluate or the drug substance. In someembodiments, the rAAV vector present in the pooled eluate or drugsubstance is produced in a vessel with a volume of 1000 L to 3000 L(e.g., about 2000 L), optionally, wherein the vessel is SUB.

In some embodiments, a pooled eluate or drug substance prepared bymethods disclosed herein is depleted of empty capsids such that emptycapsids comprise 20%+/−2% of total capsids in the pooled eluate or thedrug substance. In some embodiments, the rAAV vector present in thepooled eluate or drug substance is produced in a vessel with a volume of1000 L to 3000 L (e.g., about 2000 L), optionally, wherein the vessel isSUB.

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) by AEX comprises collecting atleast one fraction of eluate from an AEX column during an elution step(e.g., a gradient elution) and forming a pooled eluate, and optionally adrug substance, comprising rAAV vectors that may be quantified byquantitative polymerase chain reaction (qPCR) analysis of vector genomes(VG or vg). qPCR analysis may measure copies of ITR sequence, copies oftransgene sequence and/or copies of any other nucleotide sequencepresent in an intact vector genome.

An amount of VG present in a pooled eluate from an AEX column may beexpressed as a % VG column yield which refers to the amount of VGpresent in the pooled eluate collected from an AEX column (i.e., an AEXpool) as a percentage of the amount of VG present in the sample to bepurified, e.g., affinity eluate, which in some embodiments has beendiluted only, or diluted and filtered and applied to the AEX column.

A method of purifying a rAAV vector according to methods disclosedherein results in % VG column yield of 63%+/−26%. A method of purifyinga rAAV vector according to methods disclosed herein results in % VGcolumn yield of 1% to 10%, 1 to 20%, 1% to 30%, 1% to 40%, 1% to 50%, 1%to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 99%, 5% to 95%, 10% to85%, 15% to 75%, 20% to 65%, 25% to 55%, 30% to 45%, 30% to 80%, 35% to65%, 40% to 70% or 100%.

In some embodiments, purification of rAAV vector produced in a 250 L SUBby methods disclosed herein results in a % VG column yield of 40% to100%. In some embodiments, purification of a rAAV vector produced in a2000 L SUB by methods disclosed herein results in a % VG column yield of10% to 70% (e.g., 20% to 61%).

An amount of VG present in a pooled eluate from an AEX column may beexpressed as a % VG step yield which refers to the amount of VG presentin a pooled eluate collected from an AEX column (i.e., an AEX pool) as apercentage of the amount of VG present in an affinity eluate prior todilution or filtration.

A method of purifying a rAAV vector according to methods disclosedherein results in % VG step yield of 47%+/−11%. A method of purifying arAAV vector according to methods disclosed herein results in % VG stepyield of 1% to 10%, 1 to 20%, 1% to 30%, 1% to 40%, 1% to 50%, 1% to60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 99%, 5% to 95%, 10% to 85%,15% to 75%, 20% to 65%, 25% to 55%, 30% to 45%, 30% to 80%, 35% to 65%,40% to 70% or 100%.

In some embodiments, purification of rAAV vector produced in a 250 L SUBby methods disclosed herein results in a % VG step yield of 30% to 70%(e.g., 37% to 60%). In some embodiments, purification of rAAV vectorproduced in a 250 L SUB by methods disclosed herein results in a % VGstep yield of 45%+/−8%.

In some embodiments, purification of a rAAV vector produced in a 2000 LSUB by methods disclosed herein results in a % VG step yield of50%+/−13%. In some embodiments, purification of a rAAV vector producedin a 2000 L SUB by methods disclosed herein results in a % VG step yieldof 25% to 75% (e.g., 31% to 66%).

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) by AEX comprises collecting atleast one fraction of eluate from an AEX column during an elution step(e.g., a gradient elution) and forming a pooled eluate, or drugsubstance, with a reduced amount of host cell protein (HCP) as comparedto the amount of HCP in the solution. In some embodiments, a reducedamount of HCP in a pooled eluate, in at least one fraction of eluate, orin a drug substance, is lower than a level of quantification (LLOQ), asmeasured by ELISA. In some embodiments, a reduced amount of HCP in apooled eluate, in at least one fraction of eluate, or in a drugsubstance, is 10 ng to 2000 ng/1×10⁹ VG, 50 ng to 200 ng/1×10⁹ VG, 100ng to 1000 ng/1×10⁹ VG or 200 to 2000 ng/1×10⁹ VG.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3b or others) from an affinity eluate by AEX comprises reducing anamount of HCP from 1 to 500 pg/1×10⁹ VG (e.g., about 50 pg/1×10⁹) in theaffinity eluate, to an amount LLOQ in a pooled eluate, in at least onefraction of eluate, or in a drug substance, and wherein the rAAV vectoris produced in a 250 L SUB.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9,rAAV3B or others) from an affinity eluate by AEX comprises reducing anamount of HCP from 100 to 500 pg/1×10⁹ VG (e.g., about 330 pg/1×10⁹) inthe affinity eluate, to an amount LLOQ in a pooled eluate, in at leastone fraction of eluate, or in a drug substance, and wherein the rAAVvector is produced in a 2000 L SUB.

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) by AEX comprises collecting atleast one fraction of eluate from an AEX column during an elution step(e.g., a gradient elution) and forming a pooled eluate, or drugsubstance, comprising the rAAV vector and wherein the purity of the rAAVvector is at least about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% asmeasured by, e.g., analytical reverse phase HPLC, capillary gelelectrophoresis.

In some embodiments, purification of a rAAV vector produced in a 250 LSUB by methods disclosed herein results in a rAAV vector preparation(e.g., a drug substance) with a purity of 98.6%+/−0.6%. In someembodiments, purification of a rAAV vector produced in a 1000 L to 3000L (e.g., about 2000 L) vessel (e.g., SUB) by methods disclosed hereinresults in a rAAV vector preparation (e.g., a drug substance) with apurity of 99.3%+/−0.3%.

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) by AEX comprises collecting atleast one fraction of eluate from an AEX column during an elution step(e.g., a gradient elution) and forming a pooled eluate, or drugsubstance, with a percentage of HMMS of 0% to 10%. In some embodiments,a percentage of HMMS is measured by size exclusion chromatography (SEC).In some embodiments, purification of a rAAV vector produced in a 100 Lto 300 L (e.g., about 250 L) vessel (e.g., SUB) by methods disclosedherein results in a rAAV vector preparation comprising 2.6%+/−0.8% HMMSas measured by SEC. In some embodiments, purification of a rAAV vectorproduced in a 2000 L SUB by methods disclosed herein results in a rAAVvector preparation comprising 2.9%+/−0.4% HMMS.

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) by AEX comprises collecting atleast one fraction of eluate from an AEX column during an elution step(e.g., a gradient elution) and forming a pooled eluate, or drugsubstance, with about 7.0 to 25 pg residual HC-DNA/1×10⁹ VG. In someembodiments, an amount of HC-DNA is measured by qPCR. In someembodiments, purification of a rAAV vector produced in a 250 L SUB bymethods disclosed herein results in a rAAV vector preparation (e.g., apooled eluate, a drug substance) comprising 17.4+/−6.7 pg HC-DNA/1×10⁹VG. In some embodiments, purification of a rAAV vector produced in a2000 L SUB by methods disclosed herein results in a rAAV vectorpreparation (e.g., a pooled eluate, a drug substance) comprising9.3+/−1.2 pg HC-DNA/1×10⁹ VG.

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) by AEX comprises collecting atleast one fraction of eluate from an AEX column during an elution step(e.g., a gradient elution) and forming a pooled eluate, or drugsubstance, with an A260/A280 of about 1.24 to 1.32. In some embodiments,an A260/A280 is measured by size exclusion chromatography (SEC). In someembodiments, purification of a rAAV vector produced in a 250 L SUB bymethods disclosed herein results in a rAAV vector preparation (e.g., apooled eluate, a drug substance) with an A260/A280 of 1.24 to 1.32, asmeasured by SEC. In some embodiments, purification of a rAAV vectorproduced in a 2000 L SUB by methods disclosed herein results in a rAAVvector preparation (e.g., a pooled eluate, a drug substance) with anA260/A280 of 1.28 to 1.31, as measured by SEC.

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) froma solution (e.g., an affinity eluate) by AEX comprises collecting atleast one fraction of eluate from an AEX column during an elution step(e.g., a gradient elution) and forming a pooled eluate, wherein thepooled eluate is subjected to a method of filtration selected from thegroup consisting of viral filtration, ultrafiltration/diafiltration(UF/DF), filtration through a 0.2 μm filter, and a combination thereof,to produce a drug substance suitable for production of a therapeuticdrug product. In some embodiments, the drug substance is suitable foradministration to a human subject to treat a disease, disorder orcondition (e.g., Duchenne muscular dystrophy). In some embodiments, therAAV vector is an AAV9 vector.

AEX Stationary Phase Regeneration

Following elution (e.g., gradient elution) and collection of at leastone fraction of eluate comprising a full rAAV capsid from an AEX column,additional steps may be performed to prepare the column stationary phasefor further rAAV purification runs. Such steps may include, for example,sanitization, equilibration, regeneration, flush and/or storage. One ofskill in the art will understand that one or more steps may beperformed, in varying order and frequency.

A method of regenerating AEX stationary phase in a column for use infurther rAAV purification runs comprises post-use sanitizing of thestationary phase. In some embodiments, post use sanitizing of thestationary phase follows an elution step (e.g., a gradient elution). Insome embodiments, sanitizing comprises application of a solutioncomprising about 0.1 M to 1 M, about 0.2 M to 0.8 M, about 0.3 to about0.7 M or about 0.4 M to about 0.6 M NaOH to AEX stationary phase in acolumn. In some embodiments, sanitizing comprises application of asolution comprising about 0.5 M NaOH to AEX stationary phase in acolumn. In some embodiments, post-use sanitizing comprises applicationof a solution comprising about 0.5 M NaOH to AEX stationary phase in acolumn and use of an upward flow. In some embodiments, post-usesanitizing comprises application of 14.4 to 17.6 CV (e.g., about 16 CV)of a solution comprising 0.5 M NaOH to AEX stationary phase in a column.In some embodiments, post-use sanitizing comprises application of 2 to20 CV, 5 to 15 CV, 7 to 13 CV (e.g., about 5, about 7.5, about 10, about16 CV, etc.) of a solution comprising about 0.5 M NaOH to AEX stationaryphase in a column at a linear velocity of 50 to 2000 cm/hr, a flow rateof 0.2 to 3.0 L/min and/or a residence time of 2 to 15 min/CV. In someembodiments, post-use sanitizing comprises application of 14.4 to 17.6CV (e.g. about 16 CV) of a solution comprising 0.5 M NaOH to AEXstationary phase in a column at a linear velocity of 270 to 330 cm/hr(e.g., about 300 cm/hr), a flow rate of 1.5 to 2.0 L/min (e.g., about1.8 L/min) through a 6.0 to 6.6 L (e.g., about 6.4 L) column, or about314 mL/min through a 1.3 L column, and/or a residence time of 3.5 to 4.5min/CV (e.g., about 4 min/CV).

A method of regenerating a column stationary phase for further rAAVpurification runs comprises regenerating the stationary phase (in someembodiments, such a step may be referred to as a “equilibration”). Insome embodiments, regenerating a column stationary phase follows anelution step (e.g., a gradient elution). In some embodiments,regenerating comprises application of a solution comprising a salt(e.g., NaCl, sodium acetate, ammonium acetate (NH₄Acetate), MgCl₂ andNa₂SO₄) and buffering agent (e.g., Tris, BIS-Tris propane,diethanolamine, diethylamine, tricine, triethanolamine and/or bicine) toa stationary phase in a column. In some embodiments, regeneratingcomprises application of a solution comprising about 0.1 M to 5 M (e.g.,0.1 M to 4 M, 0.1 M to 3.5 M, 0.1 M to 3 M, 0.1 M to 2.5 M, 0.5 M to 4M, 0.5 m to 3.5 M, 0.5 M to 3.0 M, 0.5 M to 2.5 M, 1 M to 4 M, 1M to 3.5M, 1 M to 3 M, 1 M to 2.5 M or about 1.5 M to 2.5 M) of a salt to thestationary phase. In some embodiments, regenerating comprisesapplication of a solution comprising about 1 mM to 500 mM (e.g., 1 mM to450 mM, 1 mM to 400, 1 mM to 350 mM, 1 mM to 300 mM, 1 mM to 250 mM, 1mM to 200 mM, 50 mM to 450 mM, 50 mM to 400 mM, 50 mM to 350 mM, 50 mMto 300 mM, 50 mM to 250 mM, 50 mM to 200 mM or 50 mM to 150 mM) of abuffering agent to the stationary phase.

In some embodiments, regenerating comprises application of a solutionwith a pH of about 7.0 and 11.0 (e.g., 7.5 to 10.5, 8.0 to 10.0, 8.5 to9.5 or 8.0 to 9.0) to the stationary phase.

In some embodiments, regenerating comprises application of a solutioncomprising about 2 M NaCl, 100 mM Tris, pH 9 to AEX stationary phase ina column. In some embodiments, regenerating comprises application of asolution comprising 2 M NaCl, 25 mM Tris, pH 9 to AEX stationary phasein a column. In some embodiments, regeneration comprises application of2 to 15 CV (e.g., about 5 CV, about 10 CV) of a solution (e.g., aregeneration solution) to AEX stationary phase in a column. In someembodiments, regeneration comprises application of 4.5 to 5.5 CV (e.g.,about 5 CV) of a solution comprising 2 M NaCl, 100 mM Tris, pH 9 to AEXstationary phase in a column. In some embodiments, regenerationcomprises application of 2 to 15 CV (e.g., about 5 CV, about 10 CV) of asolution comprising 2 M NaCl, 100 mM Tris, pH 9 to AEX stationary phasein a column at a linear velocity of 100 to 2000 cm/hr, a flow rate of0.2 to 3.0 L/min and/or a residence time of 2 min/CV to 15 min/CV. Insome embodiments, regenerating comprises application of 4.5 to 5.5 CV(e.g., about 5 CV) of a solution comprising 2 M NaCl, 100 mM Tris, pH 9,to AEX stationary phase in a column at a linear velocity of 270 to 330cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5 to 2.0 L/min (e.g.,about 1.8 L/min) through a 6.0 to 6.6 L (e.g., about 6.4 L) column, orabout 314 mL/min through a 1.3 L column, and/or a residence time of 3.5to 4.5 min/CV (e.g., about 4 min/CV).

A method of regenerating a column stationary phase for further rAAVpurification runs comprises equilibration of the stationary phase (insome embodiments, such a step may be referred to as a “regenerationstep”). In some embodiments, equilibration of stationary phase in acolumn follows an elution step (e.g., a gradient elution). In someembodiments, equilibration of media in a column comprises application ofa solution comprising about 100 mM Tris, pH 9 to AEX stationary phase ina column. In some embodiments, equilibration of a column comprisesapplication of a solution comprising 20 mM Tris, pH 9 to AEX stationaryphase in a column. In some embodiments, equilibration of a columncomprises application of 2 to 15 CV (e.g., about 5 CV, 10 CV) of asolution (e.g., an equilibration solution) to AEX media in a column. Insome embodiments, equilibration of a column comprises application of 4.5to 5.5 CV (e.g., about 5 CV) of a solution comprising 100 mM Tris, pH 9to AEX stationary phase in a column. In some embodiments, equilibrationof a column comprises application of 2 to 15 CV (e.g., about 5 CV, about10 CV) of a solution comprising 100 mM Tris, pH 9 to AEX stationaryphase in a column at a linear velocity of 100 to 2000 cm/hr, a flow rateof to 3.0 L/min and/or a residence time of 2 min/CV to 15 min/CV. Insome embodiments, equilibration of a column comprises application of 4.5to 5.5 CV (e.g., about 5 CV) of a solution comprising 100 mM Tris, pH 9to AEX stationary phase in a column at a linear velocity of 270 to 330cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5 to 2.0 L/min (e.g.,about 1.8 L/min) through a 6.0 to 6.6 L (e.g., about 6.4 L) column, orabout 314 mL/min through a 1.3 L column, and/or a residence time of 3.5to 4.5 min/CV (e.g., about 4 min/CV).

A method of regenerating a column stationary phase for further rAAVpurification runs comprises post-use flushing (i.e., flushed) of thestationary phase. In some embodiments, post-use flushing of a columnfollows an elution step (e.g., a gradient elution). In some embodiments,post-use flushing of a column comprises application of water forinjection (e.g. purified water) to AEX stationary phase in a column. Insome embodiments, post-use flushing of a column comprises application of≥4.5 CV (e.g., about 5 CV) of water for injection to AEX stationaryphase in the column. In some embodiments, post-use flushing of a columncomprises application of 2 to 15 CV (e.g., about 5 CV, about 10 CV) of asolution comprising water for injection to AEX stationary phase in acolumn at a linear velocity of 100 to 2000 cm/hr, a flow rate of 0.2 to3.0 L/min and/or a residence time of 2 min/CV to 15 min/CV. In someembodiments, post-use flushing of a column comprises application of 4.5to 5.5 CV (e.g., about 5 CV) of water for injection to AEX stationaryphase in a column at a linear velocity of 270 to 330 cm/hr (e.g., about300 cm/hr), a flow rate of 1.5 to 2.0 L/min (e.g., about 1.8 L/min)through a 6.0 to 6.6 L (e.g., about 6.4 L) column, or about 314 mL/minthrough a 1.3 L column, and/or a residence time of 3.5 to 4.5 min/CV(e.g., about 4 min/CV).

A method of regenerating a column stationary phase for further rAAVpurification runs comprises applying a storage solution to thestationary phase. In some embodiments, applying a storage solution to acolumn follows an elution step (e.g., a gradient elution). In someembodiments, a storage solution comprising 16% to 20% ethanol (e.g.,about 17.5%) is applied to AEX stationary phase in a column. In someembodiments, 2 to 11 CV (e.g., about 3 CV, about 10 CV) of a storagebuffer are applied to AEX stationary phase in a column. In someembodiments, 2.7 to 3.3 CV (e.g., about 3 CV) of a storage solutioncomprising 17.5% ethanol is applied to AEX stationary phase in a column.In some embodiments, 2 to 11 CV (e.g., about 3 CV) of a storage solutioncomprising 17.5% ethanol is applied to AEX stationary phase in a columnat a linear velocity of 100 to 2000 cm/hr, a flow rate of to 3.0 L/minand/or a residence time of 2 min/CV to 15 min/CV. In some embodiments,applying a storage solution to a column comprises application of 2.7 to3.3 CV (e.g., about 3 CV) of a solution comprising 17.5% ethanol to AEXstationary phase in a column, at a linear velocity of 270 to 330 cm/hr(e.g., about 300 cm/hr), a flow rate of 1.5 to 2.0 L/min (e.g., about1.8 L/min) through a 6.0 to 6.6 L (e.g., 6.4 L) column, or about 314mL/min through a 1.3 L column, and/or a residence time of 3.5 to 4.5min/CV (e.g., about 4 min/CV).

A method of regenerating a column stationary phase for further rAAVpurification runs, the method comprising a step of: i) post-usesanitizing comprising application of 14.4 to 17.6 CV (e.g. about 16 CV)of a solution comprising about 0.5 M NaOH to the stationary phase; ii)regenerating comprising application of 4.5 to 5.5 CV (e.g., about 5 CV)of a solution comprising about 2 M NaCl, 100 mM Tris, pH 9 to thestationary phase; iii) equilibration comprising application of 4.5 to5.5 CV (e.g., about 5 CV) of a solution comprising about 100 mM Tris, pH9 to the stationary phase; iv) post-use flushing comprising applicationof 4.5 to 5.5 CV (e.g., about 5 CV) of water for injection to thestationary phase; and/or v) applying a storage solution to thestationary phase comprising application of 2.7 to 3.3 CV (e.g., about 3CV) of a storage solution comprising about 17.5% ethanol to the column;wherein at least one of steps i)-v) is performed at a linear velocity of270 to 330 cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5 to 2.0L/min (e.g., about 1.8 L/min) through a 6.0 to 6.6 L (e.g., 6.4 L)column or about 314 mL/min through a 1.3 L column, and/or a residencetime of 3.5 to 4.5 min/CV (e.g., about 4 min/CV) and wherein thestationary phase is AEX stationary phase, optionally POROS™ 50 HQstationary phase.

A method of regenerating AEX stationary phase for further rAAVpurification runs comprises application of an ethanol washout solutionto the stationary phase prior to the first step of a method of purifyinga rAAV vector (i.e., prior to sanitization, prior to equilibration,etc.). In some embodiments, an ethanol washout solution comprises about20 mM Tris, pH 9. In some embodiments, application of an ethanol washoutsolution to the column stationary phase comprises application of 8 to 12CV (e.g., about 10 CV) of a solution comprising about 20 mM Tris, pH 9to AEX stationary phase. In some embodiments, application of an ethanolwashout solution to AEX stationary phase comprises application of 8 to12 CV (e.g., about 10 CV) of a solution comprising about 20 mM Tris, pH9 to AEX stationary phase at a velocity of 100 to 1000 cm/hr (e.g.,about 600 cm/hr) and/or with a residence time of 1 to 10 min/CV (e.g.,about 1.5 min/CV).

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the disclosure. The foregoingdescription and Examples detail certain exemplary embodiments of thedisclosure. It will be appreciated, however, that no matter how detailedthe foregoing may appear in text, the disclosure may be practiced inmany ways and the disclosure should be construed in accordance with theappended claims and any equivalents thereof.

All references cited herein, including patents, patent applications,papers, textbooks, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety.

EXEMPLARY EMBODIMENTS

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

EXAMPLES Example 1: Screening of Elution Salts to Enrich for Full AAV9Vectors Via AEX Chromatography

Preparation of AEX Load for Elution Salt Screening

HEK 293 cells were grown in suspension culture and transfected with 3plasmids to produce AAV9 vector per standard methods known in the art(Grieger et al. (2016) Molecular Therapy 24(2):287-297). HEK 293 cellswere harvested, lysed, flocculated, and the resulting lysate wasfiltered. AAV9 vector was purified from the clarified lysate by affinitychromatography. An affinity column was equilibrated, loaded withclarified lysate, washed, and the purified AAV9 vector was eluted. TheAAV9 vector affinity pool (also referred to as affinity eluate) was pH4.4, with a conductivity of 5.3 mS/cm. The affinity pool was diluted7.6-fold with 20 mM Tris, pH 9, adjusted to pH 9 with 1 M Tris base, pH11, and filtered through a 0.2 μm filter. The resulting solution was pH9, with a conductivity of 1.9 mS/cm, and was loaded on to AEX columnsfor elution salt screening studies.

Elution Salt Screening Via AEX Chromatography on a 1 mL POROS™ HQ Column

Four elution salts were studied for their ability to resolve AAV9 empty(i.e., an AAV capsid that does not contain a recombinant vector genome)and full capsids (i.e., an AAV capsid that contains a recombinant vectorgenome) during AEX chromatography. Consistent with Table 1, a POROS™ 50HQ column (0.5 cm inner diameter, 5 cm bed height, 1 mL column volume)was equilibrated, loaded and washed. A 50 CV gradient was developed from0-50% B buffer, followed by a 10 CV step elution, carried out with 100%B buffer. Four B buffers were employed, with compositions of 20 mM Tris,500 mM salt, pH 9. The salts were one of NaCl, NaAcetate, NH₄Acetate, orNa₂SO₄.

TABLE 1 AEX chromatography methods for screening gradient elution saltson 1 mL POROS ™ 50 HQ column. Column Bed Load Challenge: Height: 5 cmColumn Cross Sectional Area: 0.2 cm² 9.3 × 10¹³ VG^(#)/mL resin ColumnColumn Volume: 1.0 mL Residence Diameter: 0.5 cm Column Time VelocityStep Solution Volumes (min/CV) (cm/hr) Equilibration 1 20 mM Tris, 500mM salt*, pH 9 20 0.5 611 Equilibration 2 20 mM Tris, pH 9 20 0.5 611Load Affinity Pool diluted with 20 mM 0.5 611 Tris, pH 9 and adjusted topH 9 with 1M Tris Base, pH 11 Wash 20 mM Tris, pH 9 10 0.5 611 GradientA Buffer: 20 mM Tris, pH 9 50 1.5 204 Elution B Buffer: 20 mM Tris, 500mM (0-50% B)/50 CV salt*, pH 9 Step Elution 20 mM Tris, 500 mM salt*, pH9 10 1.5 204 Regeneration 2M NaCl, 25 mM Tris, pH 9 10 0.5 611Sanitization 0.5M NaOH 10 6 51 Equilibration 20 mM Tris, pH 9 10 0.5 611Storage 20% Ethanol 10 0.5 611 *Elution salts: NaCl, NaAcetate,NH₄Acetate, Na₂SO₄ ^(#)vector genome (VG) was measured by qPCR analysisof ITR sequence.

The impact of the elution salts on the shape and A₂₆₀/A₂₈₀ ratios ofelution peaks is shown by the chromatograms of FIG. 1 . The A₂₆₀/A₂₈₀ratio provides an estimation of the percentage of AAV capsids thatcontain a recombinant vector genome (% full), with higher ratiosindicating higher % full (Sommer et al. Molecular Therapy (2003)7(1):122-128). Elution via NaCl led to a main peak with a high A₂₆₀/A₂₈₀(indicative of full vectors) and a closely joined shoulder with a lowA₂₆₀/A₂₈₀ (indicative of empty capsids). Elution via NaAcetate andNH₄Acetate generated a main peak with high A₂₆₀/A₂₈₀, and two resolvedpeaks with low A₂₆₀/A₂₈₀. These results indicate that NaAcetate andNH₄Acetate resolved empty capsids from AAV9 vectors better than NaCl.Conversely, Na₂SO₄ eluted the AAV9 material in one sharp peak withmoderate A₂₆₀/A₂₈₀, implying little separation of AAV9 vectors fromempty capsid.

One milliliter eluate fractions were collected throughout the gradientsand neutralized with 0.15 mL of 50 mM citrate, pH 3.6. Neutralizedfractions were analyzed by HPLC-SEC A₂₆₀/A₂₈₀. During the HPLC-SECmethod, absorbance was monitored at 214, 260, and 280 nm. The A₂₆₀/A₂₈₀ratio provides an estimation of the % full of AAV capsids (Sommer et al.Molecular Therapy (2003) 7(1):122-128). The AAV9 signature SEC elutionpeaks at 260 and 280 nm were integrated and the ratio of these twovalues is reported as SEC A₂₆₀/A₂₈₀ as shown in FIG. 2 . The NaAcetategradient generated elution fractions with a maximum SEC A₂₆₀/A₂₈₀ of1.27, higher than analogous maximum values for NaCl (1.23), NH₄Acetate(1.22), and Na₂SO₄ (1.15). Further, NaAcetate-based elution produced 7contiguous eluate fractions with SEC A₂₆₀/A₂₈₀≥1.19, higher thananalogous results for NaCl (3 fractions), NH₄Acetate (4 fractions) andNa₂SO₄ (0 fractions). Fractions with SEC A₂₆₀/A₂₈₀≥1.19 were pooledtogether, assayed by qPCR of the ITRs to determine vector genome (VG)yield, and analyzed by analytical ultracentrifugation (AUC) to determinedistribution of empty, full, and intermediate (AAV capsids that haveless packaged nucleic acid than full capsids and contain, for example, apartial, fragmented or a truncated vector genome and/ornon-transgene-related DNA) capsids.

The elution salt screening studies demonstrated that NaAcetateoutperformed NaCl, NH₄Acetate, and Na₂SO₄ in terms of VG yield, and %full of the recovered AAV9 vector as judged by SEC A₂₆₀/A₂₈₀ and AUC(Table 2). The NaAcetate AEX pool had an SEC A₂₆₀/A₂₈₀ of 1.24, slightlyhigher than analogous values obtained using NaCl (pool SECA₂₆₀/A_(280=1.23)) and NH₄Acetate (SEC A₂₆₀/A₂₈₀=1.21), andsignificantly higher than the Na₂SO₄ pool (SEC A₂₆₀/A₂₈₀=1.15). AUCanalysis on AEX pools implied that the NaAcetate gradient generatedslightly higher % full (43%) than the NaCl gradient (˜38%), andsignificantly higher % Full than the Na₂SO₄ gradient (20%). Notably, theNaAcetate gradient elution reduced the percentage of empty capsids (%empty) from 75% in the AEX load to 29% in the AEX pool. To betterdelineate the performance of the elution salts, NaCl, NaAcetate, andNH₄Acetate were selected to be used on a 5.1 mL POROS™ 50 HQ column,described in the section below.

TABLE 2 Results from elution salt screening studies on a 1 mL POROS ™ 50HQ column. % VG Column SEC-A₂₆₀/A₂₈₀ AUC of AEX Pool Sample/ Yield MaxFractions ≥ Pooled % % % Elution Salt (qPCR) Value 1.19 Product fullinter. empty AEX Load N/A 0.91 N/A N/A 12 13 75 NaCl 17 1.23 3 1.23 ~38~25 ~38 NaAcetate 21 1.27 7 1.24 43 27 29 NH₄Acetate 12 1.22 4 1.21 LLOQNa₂SO₄ 22 1.15 0 1.15 20 30 50 % VG Column Yield was determined as (VGin AEX pool)/(VG in AEX load); and thus, did not account for lossesincurred upon load preparation. AUC data on the NaCl gradient elutionAEX pool was inconclusive and thus is reported here as an approximation(~). LLOQ—Lower than the lower limit of quantitation.

Elution Salt Screening Via AEX Chromatography on a 5.1 mL POROS™ 50 HQColumn

Elution salts NaCl, NaAcetate, and NH₄Acetate were studied for theirability to resolve AAV9 empty capsids and full vectors during AEXchromatography. A POROS™ 50 HQ column (0.66 cm inner diameter, 15 cm bedheight, 5.1 mL column volume) was equilibrated, loaded, washed, andeluted with NaCl, NaAcetate, or NH₄Acetate gradients (Table 3). Onemilliliter elution fractions were collected throughout the gradient,neutralized with 0.15 mL of 50 mM citrate, pH 3.6, and analyzed byHPLC-SEC A₂₆₀/A₂₈₀.

NaAcetate-based elution produced 20 contiguous fractions with SECA₂₆₀/A₂₈₀>=1.19, higher than analogous results for NaCl (8 fractions)and NH₄Acetate (11 fractions). Fractions with SEC A₂₆₀/A₂₈₀≥1.19 werepooled together, assayed by qPCR of the ITRs to determine VG yield, andanalyzed by analytical ultracentrifugation (AUC) to determine % full.

TABLE 3 AEX chromatography methods for screening elution salts performedon a 5.1 mL POROS ™ 50 HQ column. Column Bed Load Challenge: Height: 15cm Column Cross Sectional Area: 0.34 cm² 3 × 10¹³ VG/mL resin ColumnColumn Volume: 5.1 mL Residence Diameter: 0.66 cm Column Time VelocityStep Solution Volumes (min/CV) (cm/hr) Sanitization 0.5M NaOH 10 2.3 389Equilibration 1 20 mM Tris, pH 9 10 0.5 611 Equilibration 2 20 mM Tris,500 mM salt*, pH 9 20 0.5 1790 Equilibration 3 20 mM Tris, pH 9 10 0.51790 Load Affinity Pool diluted with 20 mM 0.5 1790 Tris, pH 9 and 1MTris Base, pH 11 Wash 20 mM Tris, pH 9 10 0.5 1790 Gradient Elution ABuffer: 20 mM Tris, pH 9 50 1.5 597 (0-50% B)/50 B Buffer: 20 mM Tris,500 mM CV salt*, pH 9 Step Elution 20 mM Tris, 500 mM salt*, pH 9 10 1.51790 Regeneration 2M NaCl, 25 mM Tris, pH 9 10 0.5 149 Sanitization 0.5MNaOH 10 6 1790 Equilibration 20 mM Tris, pH 9 10 0.5 1790 Storage 20%Ethanol 10 0.5 597 *Elution salts: NaCl, NaAcetate, NH₄Acetate

Elution salt screening studies demonstrated that NaAcetate outperformedNaCl and NH₄Acetate in terms of the % full of the recovered AAV9 vectoras judged by SEC A₂₆₀/A₂₈₀ and AUC (Table 4). The NaAcetate AEX pool hadan SEC A₂₆₀/A₂₈₀ of 1.26, higher than analogous values obtained usingNaCl (pool SEC A₂₆₀/A₂₈₀=1.24) and NH₄Acetate (SEC A₂₆₀/A₂₈₀=1.19). AUCanalysis on AEX pools implied that NaAcetate gradients generatedslightly higher % full capsids (43%) than the NaCl gradient (39%) andthe NH₄Acetate gradient (36%).

Collectively, the AEX runs carried out at 1 mL and 5.1 mL column volumescale showed that NaAcetate resolved empty capsids from full AAV9vectors better than NaCl and NH₄Acetate. Therefore, NaAcetate wasemployed as the elution salt in further developed versions of the AEXprocess.

TABLE 4 Results from elution salt screening studies on a 5.1 mL POROS ™50 HQ column. % VG Column SEC-A₂₆₀/A₂₈₀ AUC of AEX Pool Sample/ YieldFractions ≥ Pooled % % % Elution Salt (qPCR) 1.19 Product Full Inter.Empty AEX Load N/A N/A N/A 12 13 75 NaCl 13 8 1.24 39 31 30 NaAcetate 1020 1.26 43 27 30 NH₄Acetate 7 11 1.19 36 10 54 % VG Column Yield wasdetermined as (VG in AEX pool)/(VG in AEX load); and thus, did notaccount for losses incurred upon load preparation.

Example 2: Enrichment of Full AAV9 Vectors Via AEX Chromatography with aSodium Acetate Step Elution

Based at least in part on the results in Example 1, NaAcetate wasselected to study step elution operation of an AEX chromatography columnfor separation of AAV9 empty capsids from full vectors. Affinity eluatewas generated as described in Example 1, diluted with 20 mM Tris, pH 9and 1 M Tris Base, pH 11, and filtered through a 0.2 μm filter.

Screening of Optimal NaAcetate Step Elution Conditions

For screening purposes, a nine-step wash and elution AEX method wascarried out with step increases in NaAcetate concentration in 20 mMTris, pH 9. Consistent with Table 5, a POROS™ 50 HQ column (0.66 cmID×15 cm BH; 5.1 mL CV) was equilibrated, loaded, washed, and eluted.Wash and elution buffers were formed in the FPLC system by mixing ABuffer: 20 mM Tris, pH 9, and B Buffer: 20 mM Tris, 140 mM NaAcetate, pH9. Fractions were neutralized and assayed by SEC A₂₆₀/A₂₈₀, AUC, andqPCR of the ITRs. FIG. 3 depicts the 9-step chromatogram and shows astark change in inline A₂₆₀/A₂₈₀ upon gradual increase in NaAcetateconcentration of wash and elution buffers.

TABLE 5 AEX screening method employing 9-Step NaAcetate elution (3washes and 6 elutions). Column Bed Height: 15 cm Column Cross SectionalArea: 0.34 cm² Load Challenge: Column Diameter: 0.66 cm Column Volume:5.1 mL 3.3 × 10¹³ VG/mL Linear Residence resin Velocity Time % of B StepCV (cm/hr) (min/CV) Solution Buffer* Storage Washout 10 600 1.5 20 mMTris, pH 9 0 Equilibration 20 600 1.5 20 mM Tris, 500 mM NaAcetate, pH 90 Equilibration 5 600 1.5 20 mM Tris, 7 mM NaAcetate, pH 9 5Equilibration 10 1800 0.5 20 mM Tris, pH 9 0 Sample Loading N/A 1800 0.5Affinity Pool, diluted 7.1-fold with N/A 20 mM Tris, pH 9 and 1M TrisBase, pH 11 Load Chase 10 1800 0.5 20 mM Tris, pH 9 0 pH Stabilization 2600 1.5 20 mM Tris, 7 mM NaAcetate, pH 9 5 pH Stabilization 2 600 1.5 20mM Tris, 14 mM NaAcetate, pH 9 10 pH Stabilization 2 600 1.5 20 mM Tris,21 mM NaAcetate, pH 9 15 Wash-1 2.5 600 1.5 20 mM Tris, 42 mM NaAcetate,pH 9 30 Wash-2 2.5 600 1.5 20 mM Tris, 49 mM NaAcetate, pH 9 35 Wash-32.5 600 1.5 20 mM Tris, 57 mM NaAcetate, pH 9 40 Elution-1 2.5 600 1.520 mM Tris, 64 mM NaAcetate, pH 9 46 Elution-2 2.5 600 1.5 20 mM Tris,75 mM NaAcetate, pH 9 54 Elution-3 2.5 600 1.5 20 mM Tris, 85 mMNaAcetate, pH 9 61 Elution-4 2.5 600 1.5 20 mM Tris, 95 mM NaAcetate, pH9 68 Elution-5 2.5 600 1.5 20 mM Tris, 105 mM NaAcetate, pH 9 75Elution-6 2.5 600 1.5 20 mM Tris, 109 mM NaAcetate, pH 9 78 Strip 5 6001.5 20 mM Tris, 500 mM NaAcetate, pH 9 0 Regeneration 10 1800 0.5 25 mMTris, 2M NaCl, pH 9 0 Sanitization 10 150 6 0.5M NaOH 0 Equilibration 101800 0.5 20 mM Tris pH 9 0 Storage 10 1800 0.5 20% Ethanol 0 *Wash, pHstabilization, and elution buffers were made in the FPLC system bymixing A & B Buffers; A buffer: 20 mM Tris, pH 9; B buffer: 20 mM Tris,140 mM NaAcetate, pH 9

Analytical results from the nine-step run show that empty AAV9 capsidscan be resolved from full AAV9 vectors via NaAcetate step washes andelutions (Table 6). Wash 1 and 2 selectively removed bound empty capsidsfrom the AEX column. Notably, wash 1 and 2 generated SEC A₂₆₀/A₂₈₀values of 0.58 and 0.79 respectively, and a % empty (AUC) of 98% and85%, respectively. Elution fractions 1-4 enriched for full AAV9 vector,with SEC A₂₆₀/A₂₈₀ values in the range of 1.27-1.30, and % full (AUC) inthe range of 29-53%, considerably higher than the 12% full in the AEXload. Based on these findings, a step wash and elution method based onNaAcetate was designed and is provided below.

TABLE 6 Results from NaAcetate 9-step elution studies carried out on a5.1 mL POROS ™ 50 HQ column. Fraction Load W-1 W-2 W-3 E-1 E-2 E-3 E-4E-5 Strip [NaAcetate] (mM) 20 42 49 57 64 75 85 95 105 500 Conductivity1.8 3.5 3.9 4.4 5.0 5.7 6.4 7.0 7.6 29.7 (mS/cm) SEC A₂₆₀/A₂₈₀ 0.88 0.580.79 1.16 1.27 1.29 1.30 LLOQ LLOQ 0.61 AUC % full 12 1 1 3 29 53 51 3010 2 % inter. 13 1 14 48 42 26 18 9 3 9 % empty 75 98 85 49 30 21 31 6187 89 W—Wash step; E—Elution step. LLOQ—lower than the limit ofquantitation.

Enrichment of Full AAV9 Vectors Via a Step NaAcetate Wash, ElutionMethod

Based on the results above, AEX methods with various step NaAcetate washand elution were tested for their ability to enrich for full AAV9 vectorcapsids. Consistent with Table 7, a POROS™ 50 HQ column (0.66 cm ID×15cm BH, 5.1 mL CV) was equilibrated, washed and eluted. Wash, pHstabilization and elution buffers were made in the FPLC system by mixingA Buffer: 20 mM Tris, pH 9, and B buffer: 20 mM Tris, 140 mM NaAcetate,pH 9. Across the studied methods, multiple parameters were varied,namely elution linear velocity (75-600 cm/hr), load challenge (5.1×10¹³to 1.1×10¹⁵ VG/mL resin), and concentration of NaAcetate in the washstep (57 mM or 68 mM).

The chromatogram for the step wash and elution run with a 600 cm/hrelution, 5.1×10¹³ VG/mL resin challenge, and 57 mM NaAcetate wash isgiven as in FIG. 4A and FIG. 4B. Table 8 reports results and revealsthat the developed step NaAcetate wash and elution AEX methods enrichedfor full AAV9 vector and reduced host cell protein (HCP) levels. Thedeveloped step methods increased the % full (as judged by AUC) from 18%to 40-53% and increased the SEC A₂₆₀/A₂₈₀ from 0.95 to 1.25-1.27. Inaddition to enriching for AAV9 full capsids, the developed step methodcleared high amounts of HCP and moderate amounts of host cell DNA(HC-DNA) at low column challenges. The step NaAcetate wash and elutionmethod did not provide % VG yields or % full of AAV9 as high asultracentrifugation or AEX chromatography via NaAcetate gradient elution(Examples 6, 7 and 8, below). However, the step elution approach avoidscomplex manipulations associated with ultracentrifugation.

TABLE 7 AEX methods employing Step NaAcetate elution. Runs utilizeddifferent load challenges, wash conditions, and elution residence times.Column Bed Height: 15 cm Column Cross Sectional Area: 0.34 cm² LoadChallenge: Column Diameter: 0.66 cm Column Volume: 5.1 mL 5 × 10¹³-1.2 ×10¹⁵ VG/mL Linear Residence resin Velocity Time % of B Step CV (cm/hr)(min/CV) Solution Buffer* Ethanol Washout 10 600 1.5 20 mM Tris, pH 9 0Equilibration 20 600 1.5 20 mM Tris, 500 mM NaAcetate, pH 9 0Equilibration 5 600 1.5 20 mM Tris, 7 mM NaAcetate, pH 9 5 Equilibration10 1800 0.5 20 mM Tris, pH 9 0 Sample Loading Vary 1800 0.5 AffinityPool diluted 7.1-fold with N/A 20 mM Tris, pH 9 and 1M Tris Base, LoadChase 10 1800 0.5 20 mM Tris, pH 9 0 pH Stabilization 2 600 1.5 20 mMTris, 7 mM NaAcetate, pH 9 5 pH Stabilization 2 600 1.5 20 mM Tris, 14mM NaAcetate, pH 9 10 pH Stabilization 2 600 1.5 20 mM Tris, 21 mMNaAcetate, pH 9 15 Wash 5 600 1.5 20 mM Tris, 57 mM NaAcetate, pH 9 or41 or 48 20 mM Tris, 67 mM NaAcetate, pH 9 Elution 5 600, 150, 1.5, 6,20 mM Tris, 100 mM NaAcetate, pH 9 72 or 75 or 12 Strip 5 600 1.5 20 mMTris, 500 mM NaAcetate, pH 9 100 Regeneration 10 1800 0.5 25 mM Tris, 2MNaCl, pH 9 0 Sanitization 10 150 6 0.5M NaOH 0 Equilibration 10 1800 0.520 mM Tris, pH 9 0 Storage 10 1800 0.5 20% Ethanol 0 *Wash, pHstabilization and elution buffers were made in the FPLC system by mixingA & B Buffers; A buffer: 20 mM Tris, pH 9; B buffer: 20 mM Tris, 140 mMNaAcetate, pH 9

TABLE 8 Results from AEX runs carried out with NaAcetate step elutionmethods carried out on a 5.1 mL POROS ™ 50 HQ column. Process InputsProcess Performance: Analysis of Step Conc. of Elution % VG ElutionFraction NaAcetate Challenge Res. Column % Capsid Species A₂₆₀/ ng HCP/ng DNA/ (mM) in (VG/mL Time Yield (AUC) A₂₈₀ 1 × 10¹⁴ 1 × 10¹⁴ Washresin) (min) (qPCR) Full Inter. Empty (SEC) VG VG AEX Starting Material18 9 73 0.95 19157 1070 57 5.1 × 10¹³ 1.5 14 50 28 22 1.27 LLOQ 387 675.1 × 10¹³ 1.5 7 38 39 23 1.27 LLOQ 457 57 2.5 × 10¹⁴ 1.5 28 53 18 291.26 LLOQ 153 57 5.7 × 10¹⁴ 1.5 31 49 20 31 1.26 94 783 57 1.1 × 10¹⁵1.5 26 50 16 34 1.27 95 328 57 1.6 × 10¹⁴ 6.0 33 49 25 26 1.27 LLOQ 103857 1.6 × 10¹⁴ 12.0 43 40 36 24 1.25 170 972 % VG Column Yield wasdetermined as (VG in AEX pooled eluate)/(VG in AEX load); and thus, didnot account for losses incurred upon load preparation.

Example 3: Screening of Methods to Prepare AAV9 Affinity Eluates for AEXChromatography

One embodiment of large-scale downstream processing of AAV9 involvescell lysis, filtration, and affinity chromatography, with productelution at low pH and moderate conductivity. Studies on viral proteinsof various AAV serotypes report calculated isoelectric points of ˜6.2and ˜5.8 for empty and full AAV9 capsids, respectively (Venkatakrishnanet al., J. Virology (2013) 87.9:4974-4984). Screening of variousconditions revealed that AAV9 only binds to AEX resins at relativelyalkaline, low conductivity environments (data not shown). Therefore,preparation of acidic AAV9 affinity eluates for AEX chromatographyrequires raising the pH and lowering the conductivity of the vectorcontaining buffer. This process traverses through the AAV9 isoelectricpoint, which is an unstable transition that can lead to vector loss.

This Example details various approaches to process acidic affinity poolsinto AEX chromatography loads. Load preparation methods of dilution,in-line mixing (FIG. 5 ), and tangential flow filtration (TFF) werestudied. The results show that processing AAV9 affinity eluates into AEXchromatography loads with high product yield and low aggregationrequired specialized development of novel and inventive processes andprocedures.

Dilution Methods to Prepare AAV9 Affinity Pools for AEX Chromatography

Affinity elution pools have a pH of about 3.8-4.4, a conductivity ofabout 5.5-6.5 mS/cm, and comprise 7×10¹³-1.4×10¹⁴ AAV9 VG/mL. Affinitypools may be prepared for AEX chromatography as described in Example 1,that is, by dilution of the pool with alkaline buffers. Consistent withTable 9, AAV9 affinity pools were diluted with combinations of alkalinebuffers in PETG vessels to raise pH and decrease conductivity. Resultingsolutions were passed through 0.2 μm filters pre-wetted with diluentbuffers. The diluted samples were 0.2 μm filtered to mimic large scaledownstream processing, in which filters are placed at the inlet ofchromatography columns. The resulting filtrates were pH 8.7-9.0, withconductivity in the range of 1.8-2.1 mS/cm, conditions that would enablehigh binding of AAV9 to AEX resins. Samples were taken after dilution,and after filtration, and assayed for VG titer by qPCR of the ITRs.

Table 9 reports results and reveals that high amounts of AAV9 were lostduring dilution and filtration. Sequential dilution with 20 mM Tris, pH9 and pH adjustment with 1 M Tris Base, pH 11, followed by 0.2 μmfiltration resulted in 39% VG loss. Reversing the order of thesediluents led to a similar result and a post filtration vector loss of37%. Larger dilutions led to higher amounts of VG loss. A 25-folddilution with 100 mM Tris, pH 9, followed by pH adjustment with 1 MTris, pH 9 yielded a post filtration VG yield of only 36%. 15-folddilution with the same buffers gave post filtration yields of 65%.

In order to reduce non-specific binding of AAV9 to the surfaces ofdilution vessels and filters, 0.01% (v/v) poloxamer 188 (P188) was addedto dilution buffer 100 mM Tris, pH 9. This approach only marginallyincreased the post filtration VG yield from 65% to 74%, without and with0.01% P188, respectively. These dilution techniques afforded % VG yieldsthat were lower than desired, so additional buffer exchange techniqueswere investigated.

TABLE 9 Screening of methods to prepare AAV9 Affinity Eluates for AEXchromatography. % VG Yield (qPCR) Post Post Buffer Buffer Buffer FinalMetrics Exchange Exchange Exchange Dilution Cond. (Dilution and 0.2 μmMethod Buffer 1 Buffer 2 Factor pH (mS/cm) or TFF) Filtration Dilution,pH 20 mM 1M Tris 8.6 8.9 1.8 70 61 Adjustment Tris, pH 9 Base, pH 11 pH1M Tris 20 mM 7.6 9.0 1.9 63 63 Adjustment, Base, pH 11 Tris, pH 9Dilution 25- Fold 100 mM 1M Tris, 25 9.0 2.0 44 36 Dilution Tris, pH 9pH 9 15- Fold 100 mM 1M Tris, 15 8.9 2.1 70 65 Dilution Tris, pH 9 pH 915- Fold 100 mM 15 8.8 2.0 76 74 Dilution, Tris, 0.01% — 0.01% P188, pH9 P188 In-Line 100 mM N/A 15 8.7 2.1 79 N/A Mixing Tris, pH 9 TFF #1 100mM N/A 1.7 9.0 2.0 66 65 Tris, 2 mM MgCl2, 0.01% P188 pH 9.0 TFF #2 with100 mM 100 mM 1.7 9.0 2.0 78 78 Arginine Tris, 500 mM Tris, 2 mM BufferArginine, MgCl2, 2 mM 0.01% MgCl2, P188 0.01% pH 9.0 P188, pH 9.0 % VGYield was determined as (VG in AEX load)/(VG in Affinity Pool); 1M TrisBase, pH 11, and 1M Tris, pH 9 were used for pH adjustment, and employedat dilution factors of less than 1-fold relative to the affinity pool.

In-Line Dilution to Prepare AAV9 Affinity Pools for AEX Chromatography

In-line dilution of AAV9 affinity pools was investigated to generate AEXloads while reducing surface area the vector was exposed to and reducingthe amount of time the vector was exposed to said surfaces. The in-linedilution apparatus is shown in FIG. 5 .

Three pieces of platinum cured silicone tubing were joined together by aY-connector. A peristaltic pump delivered 100 mM Tris, pH 9 to theY-connector at flow rate of 3.5 mL/min. A second peristaltic pumpdelivered AAV9 affinity pool to the Y-connector at a flow rate of 0.25mL/min. The ratio of these flow rates (14 parts diluent to 1-partaffinity pool) was selected to achieve an 15-fold dilution and enabledirect comparison to similar dilution factors (Table 9 and Table 12).The joined fluids passed through platinum cured silicone tubing with aninner diameter of 0.16 cm and a length of 100 cm. The mixing tubedimensions were designed based on buffer mixing studies that showedthese conditions achieved a stable, well blended solution.

The in-line mixed solution was collected, neutralized with 250 mM sodiumcitrate, pH 3.5, and analyzed by qPCR of the ITRs. 79% of the of the VGpumped into the apparatus was recovered at the outlet of the mixingtubing. While this result represented a slight improvement in % VG yieldcompared to batch dilution experiments, the yield was still lower thandesired. Therefore, further AEX load preparation experiments werecarried out via tangential flow filtration.

Tangential Flow Filtration (TFF) to Prepare AAV9 Affinity Pools for AEXChromatography

In order to avoid VG losses incurred during AEX load preparation, TFFwas used to keep the VG concentration high and temporarily incorporatearginine into the vector containing solution. Consistent with Table 10,two TFF runs were carried out with fresh 20 cm² mPES hollow fibermembranes. The membrane was equilibrated, loaded with AAV9 affinitypool, and diafiltered against 150 mM acetate, 100 mM glycine, 25 mMMgCl₂, pH 4.2, the same buffer of the AAV9 affinity pool. At the end ofeach step, the TFF system was paused and the retentate vessel wassampled. Samples were analyzed for VG titer by qPCR of the ITRs andresults are shown in Table 9 and Table 10.

In run #1, diafiltration directly from the affinity elution pool analogbuffer into 100 mM Tris, 2 mM MgCl₂, 0.01% P188, pH 9 resulted in a 66%VG yield. In run #2, addition of 500 mM arginine (a co-solvent known toreduce protein aggregation (Arakawa et al. Biophysical Chemistry (2007)127(1): 1-8)) into diafiltration buffer 2 improved the VG yield to 93%compared to the 66% yield obtained in the absence of arginine. However,removal of arginine from the system in run #2 incurred significantlosses and a VG yield of 78%. 500 mM arginine was not included in thefinal diafiltration buffer because it would generate AEX loads with highconductivity (˜20 mS/cm), which would interfere with binding to AEXresins. However, this finding encouraged the study of other amino acidcosolvents that can stabilize AAV9, but with lower solventconductivities at alkaline pH. These studies are described in Example 6.

TABLE 10 Tangential flow filtration to prepare AAV9 affinity eluates forAEX chromatography. Membrane Challenge: Membrane: 300 kDa 1.2 × 10¹⁴VG/cm² Transmembrane Pressure: mPES Hollowfiber, 20 cm² Permeate Flux: 5PSI Inlet Pressure: 5-10 PSI 75 liters/m²/hr Diafiltration Precip- % VGRun Step Buffer Volumes itation? Yield TFF Membrane 150 mM Acetate, 100mM 2.0 N/A N/A #1 Equilibration Glycine, 25 mM MgCl₂, pH 4.2 Sample LoadAffinity Eluate N/A no 100%  Diafiltration 1 150 mM Acetate, 100 mM 2.0no 80% Glycine, 25 mM MgCl₂, pH 4.2 Diafiltration 2 100 mM Tris, 2 mMMgCl₂, 2.5 yes 66% 0.01% P188 pH 9.0 Retentate 0.2 N/A N/A N/A 65% umFiltration #2) Membrane 150 mM Acetate, 100 mM 2.0 N/A N/A TFFEquilibration Glycine, 25 mM MgCl₂, pH 4.2 with Sample Load AffinityEluate N/A no 100%  Arginine Diafiltration 1 150 mM Acetate, 100 mM 2.0no 95% Buffer Glycine, 25 mM MgCl₂, pH 4.2 Diafiltration 2 100 mM Tris,500 mM Arginine, 1.5 no 93% 2 mM MgCl₂, 0.01% P188, pH 9.0 Diafiltration3 100 mM Tris, 2 mM MgCl₂, 2.5 yes 78% 0.01% P188 pH 9.0 Retentate 0.2N/A N/A N/A 78% um Filtration

Dynamic Light Scattering Analysis of AAV9 Affinity Pools Upon Dilutionwith 100 mM Tris, pH 9

Dynamic light scattering (DLS) was used to measure the Z-average (Z-AVG)as an estimate of capsid aggregation in AAV9 affinity pools diluted with100 mM Tris, pH 9. The Z-average is a reliable measure of the averagesize of particles in solution. As shown in Table 11, AAV9 affinity poolswere diluted (0 to 30-fold) with 100 mM Tris, pH 9 in polypropylenetubes and immediately analyzed by DLS. For each dilution factor, aseparate experiment was carried out with fresh AAV9 affinity pool in anew polypropylene tube. Once DLS analysis was complete the solution wasmeasured for pH and conductivity.

The results are summarized in Table 11, graphed in FIG. 6 , and showthat dilution of AAV9 affinity pools led to aggregation and an increasedZ-average. The undiluted AAV9 affinity pool had a pH of 4.1,conductivity of 6.0 mS/cm, a Z-average of 15 nm, and no aggregation.Two-fold dilution with 100 mM Tris, pH 9 increased solution pH to 7.2,maintained conductivity at 5.8 mS/cm, led to a 5-fold increase inaggregation and a 77 nm Z-average, compared to the AAV9 affinity pool.This result implied that raising solution pH through the estimated AAV9isoelectric point ((calculated to be ˜5.8 and ˜6.2 for full and emptyAAV9 capsids, respectively (Venkatakrishnan et al. (2013) J. Virology87(9):4974-4984)) destabilized the vector, leading to product loss.Dilution factors of 5- and 10-fold led to aggregation and very highZ-averages of 395 nm, and 221 nm, respectively. Larger dilution factorsin the range of 15- to 30-fold displayed Z-averages of 46-66 nm andaggregation.

Collectively, the results given in Table 9 and Table 10 revealed thathigh amounts of AAV9 vector were lost during AEX load preparation. Loadpreparation methods of TFF at high VG concentration, in-line dilution,and dilution in the presence of 0.01% P188 were unable to prevent VGlosses. Data in Table 11 demonstrated that the mechanism behind the VGlosses was aggregation. Based on this observation, a series of dilutionexperiments were carried out with diluents that could prevent theaggregation and are described in Example 6.

TABLE 11 pH, conductivity, Z-Average, and Aggregation of AAV9 affinitypool diluted with 100 mM Tris, pH 9. Dilution Conductivity Z-AverageFactor pH (mS/cm) (nm) Aggregation # 0 4.1 6.0 15 − 2 7.2 5.8 77 + 5 8.53.4 395 + 10 8.7 2.3 221 + 15 8.8 2.2 66 + 20 8.9 1.9 53 + 25 8.9 1.846 + 30 8.9 1.8 52 + # aggregation present (+) or aggregation notpresent (−)

Example 4: Screening of Diluent Co-Solvents to Prepare AAV9 AffinityEluates for AEX

AAV9 affinity eluates were diluted 15-fold with various diluentco-solvents to identify conditions that maximized % VG yield during AEXload preparation. The screened cosolvents included detergents,iodixanol, glycerol, magnesium chloride, and amino acids. In order tostudy the effect of dilution alone, without altering pH or conductivity,AAV9 affinity eluate was diluted with affinity eluate pool buffer,namely 150 mM acetate, 100 mM glycine, 25 mM MgCl₂, pH 4.2. 14 mL ofdiluent was added to polypropylene tubes, followed by 1 mL of AAV9affinity eluate. The resulting solution was gently mixed via end-to-endagitation, measured for pH and conductivity, and a pre-filtration samplewas taken. The diluted sample was then filtered through a 0.2 μm filterpre-wetted with diluent. Post dilution and post filtration samples wereneutralized with 250 mM sodium citrate, pH 3.5. Neutralized samples wereanalyzed by dynamic light scattering to estimate particle Z-AVG andrelative amounts of aggregation and analyzed by qPCR of the ITRs todetermine VG titer.

Results of diluent co-solvent screening revealed some cosolvents reducedaggregation, maintained Z-AVG near 30 nm, and increased % VG yield,compared to baseline diluent 100 mM Tris, pH 9 (Table 12). The undilutedAAV9 affinity elution pool had a Z-AVG of 29 nm, with apparently noaggregation. Dilution of the AAV9 affinity pool with affinity eluatepool buffer resulted in no increase in Z-AVG, no aggregation, but only69% VG yield. This data suggested that aggregation did not occur at theconditions of the affinity pool (pH 4.2, 7 mS/cm), but VG loss occurredvia non-specific binding to increased surface area (by dilution).

In agreement with results from Example 3 (above), dilution of the AAV9affinity pool with 100 mM Tris, pH 9 led to increased Z-AVG, highaggregation, and VG yields of only 59%. Addition of 0.01-1% P188 to 100mM Tris, pH 9 did not significantly improve performance, with similarZ-AVG and aggregation, compared to the baseline buffer. Dilution of AAV9affinity eluate with 50 mM arginine, 2 mM MgCl₂, 0.1% P188, 100 mM Tris,pH 9 led to a 33 nm Z-AVG, no aggregation, and ˜80% VG yield, but led toa conductivity of 4.5 mS/cm, which would interfere with binding of AAV9to AEX resins. Multiple histidine-containing diluents provided desirableresults. For example, dilution of AAV9 with 200 mM Histidine, 200 mMTris, 10 mM MgCl₂, 25% lodixanol, pH 8.8 led to a 99% VG yield.lodixanol is strongly UV active and would interfere with UV readings inchromatography systems, so this and similar buffers were not employed inAEX runs.

Importantly, 15-fold dilution of the AAV9 affinity eluate with 0.5%P188, 200 mM histidine, 200 mM Tris, pH 8.8, followed by filtrationthrough a pre-wetted filter resulted in a 35 nm Z-AVG, and 101% VGyield. The resulting diluted, filtered solution was pH 8.8, with aconductivity of 2.5 mS/cm, conditions that were likely amenable tobinding to AEX resins. Therefore, this dilution scheme was optimized inthe example that follows.

Studies of AAV2 aggregation involved screening of various cosolvents viaa dilution stress test in combination with DLS and found that AAV2aggregation was prevented by dilution into buffers that containedvarious salts at ionic strength 200 mM (Fraser et al. Molecular Therapy(2005) 12(1):171-178). Similar approaches to prepare AAV9 for AEXchromatography were not applicable to the present Example becausesolutions with ionic strengths 200 mM reduced vector binding to AEXresins (data not shown). Interestingly, addition of the amino acidshistidine, arginine, or glycine to the diluent did not inhibit AAV2aggregation (Fraser et al. Molecular Therapy (2005) 12(1):171-178).

In this Example, mixtures of histidine, arginine, and glycine, incombination with MgCl₂, P188, and/or glycerol reduced AAV9 aggregation,but only afforded % VG yields of 69-80% (see diluent results R2, H2, H3in Table 12). High % VG yields were only achieved when histidine wasemployed in synergy with detergents P188 and Triton X-100 (see diluentresults H7 and H8 in Table 12). These results indicate that high VGyielding diluent cosolvents for AAV capsid (e.g., AAV9) AEX loadpreparation should contain detergent to reduce non-specific binding tosurfaces (e.g. dilution vessel and filter) and employ histidine orsimilar moieties to modulate charge interactions and/or hydrogen bondingbetween AAV capsid particles (e.g., AAV9 vector particles).

TABLE 12 Screening of diluent cosolvents for AEX load preparation ofAAV9 vector capsids. AAV9 affinity eluates were diluted 15-fold withdiluents, filtered, analyzed by dynamic light scattering to determineZ-average (Z-AVG) as an estimate of capsid aggregation, and analyzed byqPCR to determine % VG Yield. % VG Diluent Buffer Matrix/ Cond. Z-aver.Aggregation Yield Code Cosolvent Subset pH (mS/cm) (nm) (−, +) (qPCR) AEAffinity Eluate AE1 Affinity Elution Pool- No Dilution 4.2 6.0 29.1 −100 AE2 150 mM Acetate, 100 mM Glycine, 25 4.2 7.3 29.4 − 69 mM MgCl₂,pH 4.2 BL Baseline, 100 mM Tris, pH 9 BL1 100 mM Tris, pH 9.0 8.8 2.074.2 + 59 PO Baseline + P188 PO1 100 mM Tris, 0.01% P188, pH 9.0 8.8 2.354.8 + NT PO2 100 mM Tris, 0.1% P188, pH 9.0 8.8 2.2 60.8 + NT PO3 100mM Tris, 1% P188, pH 9.0 8.8 2.1 87.2 + NT R, G Arginine and Glycine R15 mM Arginine, 2 mM MgCl₂, 0.1% 8.9 2.8 68.3 + 81 P188, 100 mM Tris, pH8.9 R2 50 mM Arginine, 2 mM MgCl₂, 0.1% 9.0 4.5 33.0 − 80 P188, 100 mMTris, pH 9.0 R2 500 mM Arginine, 2 mM MgCl₂, 0.1% 9.1 20.6 16.2 + 88P188, 400 mM Tris, pH 9.1 G1 200 mM Glycine, 5 mM MgCl₂, 200 8.9 2.0 N/ANR 70 mM Tris, pH 8.9 H Histidine H1 200 mM Histidine, 200 mM Tris, pH8.9 2.1 31.3 + NT 8.9 H2 200 mM Histidine, 200 mM Tris, 5 mM H2 200 mMHistidine, 200 mM Tris, 5 mM 8.9 2.6 30.4 − 69 MgCl₂, pH 8.9 H3 200 mMHistidine, 200 mM Tris, 5 mM 8.9 2.3 31.8 − 74 MgCl₂, 5% Glycerol, pH8.9 H4 200 mM Histidine, 250 mM Tris, 10 8.9 1.8 19.6 + 76 mM MgCl₂, 25%Glycerol, pH 8.9 H5 200 mM Histidine, 200 mM Tris, 5 mM 8.8 2.4 12.9 +74 MgCl₂, 5% Iodixanol, pH 8.8 H6 200 mM Histidine, 200 mM Tris, 10 8.82.5 3.5 NR 99 mM MgCl₂, 25% Iodixanol, pH 8.8 H7 200 mM Histidine, 200mM Tris, 0.5% 8.8 2.5 6.5 NR 105 Triton X-100, pH 8.9 H8 200 mMHistidine, 200 mM Tris, 0.5% 8.8 2.5 35.0 NR 101 P188, pH 8.8 NR—valuenot reported due to interference in DLS readout. NT—experiment notconducted.

Example 5: Optimization of Diluent Co-Solvents to Prepare RecombinantAAV9 Vector Affinity Eluates for AEX Chromatography

The concentration of P188 in the diluent, and the resulting conductivityof the diluted sample were optimized to achieve maximum recovery of AAV9vector. Diluent P188 concentrations of 0.01%, 0.05%, 0.2%, and 0.5% weretested in concert with diluted sample conductivities of 2, 2.5, and 3mS/cm. In order to achieve the varying conductivities, varying dilutionfactors were employed. Diluted AAV9 vector affinity eluates were passedthrough 0.2 μm filters that were pre-wetted with each correspondingdiluent. The resulting filtrates were assayed by qPCR of the transgeneto determine VG titer, and results are shown in Table 13 and FIG. 7A andFIG. 7B. The data shown in FIG. 7A and FIG. 7B demonstrates that 0.5%P188 is required to achieve maximal VG yields and that use of lowerconcentrations (i.e., less than 0.5%) of P188 lead to lower VG yields.Neither final conductivity (or by association, dilution factor)significantly influenced the % VG yield within the ranges studied.

TABLE 13 Optimization of P188 concentration, dilution factor, and finalconductivity. % VG Yield Conductivity Dilution Post Dilution P188 (%)(mS/cm) Factor and Filtration 0.2 3.0 5x 93% 0.2 2.5 9x 107%  0.01 3.05x 64% 0.05 2.5 9x 88% 0.5 2.0 25x  107%  0.01 2.0 25x  79% 0.2 2.5 9x83% 0.2 2.0 25x  87% 0.5 3.0 5x 110%  0.5 2.5 9x 90% 0.01 2.5 9x 82% 0.52.5 9x 101% 

Example 6: Enrichment of Full AAV9 Via Optimized Dilution and AEXChromatography Techniques

The top performing diluent from Examples 4 and 5 was combined with thetop performing elution salt from Example 1 to form an optimized AEXchromatography method capable of enriching for full AAV9 vector capsidswith high % VG yields.

AAV9 affinity eluate was diluted 15-fold with a novel buffer comprisingan amino acid and detergent cosolvent (200 mM Histidine, 200 mM Tris,0.5% P188, pH 8.9) and filtered with a 0.2 μm filter. The 15-folddilution is lower than the dilution used in other methods (see, US2019-0002841; US 2019-0002842; US 2018-0002844), is easier to implementin large scale manufacturing and results in high VG yields.

The resulting filtrate was pH 8.8, with a conductivity of 2.3 mS/cm.Consistent with Table 14, the filtrate was loaded onto a POROS™ 50 HQcolumn, eluted via a sodium acetate gradient, and fractions equivalentto 0.39 of a CV were collected during the gradient elution.

Load and chromatographic fractions were neutralized with 250 mM sodiumcitrate, pH 3.5, and assayed by SEC A₂₆₀/A₂₈₀, AUC, and qPCR of theITRs. To test the reproducibility of the AEX process, a second run wascarried out using the same materials and methods of the first run. TheAEX chromatogram of the first run is depicted in FIG. 8A and FIG. 8B.SEC A₂₆₀/A₂₈₀ of the gradient elution fractions is provided in Table 15and shows that the optimized AEX method enriched for full AAV9 vector ascompared to the load. The percentage of full, intermediate and emptycapsid in the affinity pool (which was loaded on the column), theflow-through fraction and elution pools was determined using analyticalultracentrifugation. The data is provided in Table 16 and shows that theoptimized AEX method enriched for the percentage of full AAV9 vectorcapsid while affording high % VG yield.

TABLE 14 Optimized AEX chromatography method performed on a 5.1 mlPOROS ™ 50 HQ column. Column Bed Load Challenge: Height: 15 cm 1.7 ×10¹⁴ VG/mL of Resin (run 1); Column Column Cross Sectional or 7.5 × 10¹³VG/mL of Resin (run 2) Diameter: 0.66 cm Area: 0.34 cm² Column ResidenceStep Volume = 5.1 mL Time Flow Rate Description Solution Description CV(min/CV) (mL/min) Equilibration 1 500 mM sodium acetate, 100 mM 5 4 1.28Tris, 0.01% P188, pH 8.9 Equilibration 2 0.5% P188, Tris, pH 9.0 (run 1)or 5 4 1.28 200 mM Histidine, 200 mM Tris, 0.5% P188, pH 8.8 (run 2)Sample Loading Affinity Eluate, diluted 15X with 0.5% N/A 4 1.28 P188,200 mM Histidine, 200 mM Tris pH 8.8; filtered through 0.2 μm filterEquilibration 3 100 mM Tris, 0.01% P188, pH 8.9 5 4 1.28 Sodium AcetateA - 100 mM Tris, 0.01% P188, pH 8.9 20 11 0.47 Gradient Elution B - 500mM NaAcetate, 100 mM Tris, 0.01% P188, pH 8.9 Gradient is run from0-100% B over 20 CV Collect 2.0 mL fractions (0.39 of a CV) throughoutelution Gradient Hold 500 mM sodium acetate, 100 mM 5 11 0.47 Tris,0.01% P188, pH 8.9 Sanitization 0.5M NaOH 5 11 0.47 Regeneration 2MNaCl, 100 mM Tris pH 9 5 11 0.47 Equilibration 4 100 mM Tris, pH 9 5 110.47 Storage 17% Ethanol 5 11 0.47

TABLE 15 SEC A₂₆₀/A₂₈₀ analysis of fractions from two replicates of theoptimized AEX process. Run Load F/T 1 2 3 4 5 6 7 8 9 10 1 0.98 0.701.23 1.28 1.30 1.31 1.31 1.28 1.24 1.17 1.12 1.06 2 0.97 0.64 1.23 1.281.29 1.30 1.28 1.25 1.20 1.14 1.09 1.03 Run Load F/T 11 12 13 14 1 0.980.70 1.04 1.01 0.96 0.92 2 0.97 0.64 0.96 0.93 0.85 0.86 F/T:flow-through (unbound fraction); Gradient elution fractions are numbered1-14.

TABLE 16 Performance of the optimized AEX method as judged by analysisof process intermediates and chromatographic fractions. AnalyticalUltracentrifugation % VG A₂₆₀/A₂₈₀ (AUC) Yield (HPLC- % Sample Name(qPCR) SEC) % Full Intermediate % Empty Affinity Pool 100%  0.99 18% 15%67% Run 1 Diluted Affinity Pool 87% 0.98 — — — Filtered AEX Load 93%0.98 — — — Flow-Through 10% 0.70  1% 14% 85% Broad Pool, Fractions 53%1.27 47% 24% 29% 1-8 Narrow Pool, Fractions 36% 1.29 55% 24% 21% 2-6Second Peak, 20% 1.07 28% 10% 63% Fractions 8-13 Run 2 Broad Pool,Fractions 52% 1.25 — — — 1-8 Narrow Pool, Fractions 39% 1.28 50% 22% 28%2-6 Second Peak, 11% 1.03 — — — Fractions 8-13 % VG yields werecalculated as (VG amount at each step or fraction)/(VG amount in theAffinity Pool), and thus, accounts for losses upon load preparation. Adash (—) indicates assay not performed.

Dilution and filtration of the AAV9 affinity eluate resulted in a 93% VGyield, providing confirmation of results in Examples 3 and 4. Theflow-through fraction contained 10% of the VG from the affinity poolstarting material, had an SEC A₂₆₀/A₂₈₀ of 0.70, and had a capsidpopulation of 1% full, 14% intermediate, and 85% empty. This resultindicates the developed AEX method partitions some empty capsids throughthe column, facilitating further enrichment by sodium acetate gradientelution.

For both AEX runs, three virtual elution pools were formed, namely abroad pool that consisted of fractions 1-8, a narrow pool comprised offractions 2-6, and a second peak pool made from fractions 8-13. Thesecond peak pool had a 20% VG yield and was comprised of mostly emptycapsids. The broad pool contained 53% VG yield, had an average SECA₂₆₀/A₂₈₀ of 1.26, and 47% full AAV9 vector capsids, thus representing a2.6-fold enrichment in % full. The narrow elution pools contained anaverage of 38% VG yield, a 1.28-1.29 SEC A₂₆₀/A₂₈₀ ratio, and an averageAAV9 vector capsid population of 53% full, 23% intermediate and 24%empty. Thus, the optimized AEX method enriched for full AAV9 vector2.9-fold, and depleted empty capsids 2.8-fold.

Results obtained from the narrow pool represented a desirable balancebetween % full and % VG yield. Therefore, to obtain similar results inthe majority of examples that follow, we adopted a fraction poolingthreshold based on SEC A₂₆₀/A₂₈₀≥1.25 (similar to the 1.25 minimum valueobtained in narrow pool fractions 2-6, Table 16). Thus, in the 8 out of10 large scale AEX runs in the examples that follow, fractions with SECA₂₆₀/A₂₈₀≥1.25 were included in pools and all fractions with SECA₂₆₀/A₂₈₀≤1.24 were excluded.

The data and strategy above illustrate a powerful characteristic of theoptimized AEX method: flexibility in pooling strategy. Utilizing highresolution gradient elution chromatography in coordination with SECA₂₆₀/A₂₈₀ analysis of collected fractions ensures high % full of therecovered product, regardless of slight changes in feed stream orprocess operation.

In contrast to other chromatographic methods developed for thepurification of recombinant AAV vectors and in particular, theseparation of empty capsids from full AAV vectors (US 2019-0002841; US2019-0002842; US 2018-0002844), the present disclosed method utilizes alower dilution factor, a dilution buffer that contains histidine, asteeper elution gradient, NaAcetate as the elution salt and lessalkaline conditions (pH 9). The method disclosed herein, and exemplifiedin Examples 1-9, also differs from other reported methods, such as thoseof Tomono et al. (Molec. Ther. Meth. Clin. Dev. (2018) 11:180-190), inthat the disclosed methods do not utilize an AEX load preparation with aconductivity of about 7 mS/cm, do not utilize ammonium sulfateprecipitation and do not utilize size exclusion chromatography. Thenovel and inventive methods disclosed herein can be implemented at alarge scale and produce a high yield of VG from an affinitychromatography eluate.

To further illustrate the flexibility and robustness of the optimizedAEX method, Example 7 tested feed streams with varying % full vectorcapsid.

Example 7: Effect of the Percentage of Full Capsids in an AffinityEluate on Performance of the Optimized AEX Process

The optimized AEX method was tested for its ability to enrich for fullAAV9 vectors from feed materials with varying percentages of AAV9 emptycapsids. Briefly, HEK293 cells were grown in suspension culture andtransfected with an adenoviral helper plasmid and a Rep2Cap9 plasmid (aplasmid comprising the transgene cassette was not included). Cells werecultured for three days post transfection, harvested, lysed,flocculated, depth filtered and absolute filtered. Affinitychromatography was performed on the resulting filtrate to generate anaffinity pool containing AAV9 particles that did not contain a vectorgenome (null transfection AAV9 affinity eluate, referred to herein asnull capsids). In order to generate AEX starting materials with varyingpercentages of full capsids, null transfection AAV9 affinity eluate wasmixed with a standard AAV9 affinity eluate at volumetric ratios of 0%,20%, 40%, 60%, 80%, and 100% null capsids.

The mixtures were diluted 15-fold with 200 mM histidine, 200 mM Tris,0.5% P188, pH 8.8, and 0.2 μm filtered to generate AEX loads that werepH 8.8, with a conductivity of 2.6 mS/cm. Consistent with Table 17, theoptimized AEX method was performed on the 6 loads that contained varyingpercentages of null-transfection generated capsids. For each of the 6AEX runs, a 6.67 mL POROS™ 50 HQ column was uniformly challenged with1.5×10¹⁵ total viral particles (VP), or 2.2×10¹⁴ VP/mL resin.Chromatographic load, flow-through, and elution fractions wereneutralized with 250 mM sodium citrate, pH 3.5, and analyzed by SECA₂₆₀/A₂₈₀.

SEC A₂₆₀/A₂₈₀ data is reported in Table 18 and FIG. 9 , and show thatthe optimized AEX method generated individual elution fractions with SECA₂₆₀/A₂₈₀≥1.25 when 0%, 20%, and 40% null capsid transfection startingmaterials were employed. Column loads that contained 0%, 20%, and 40%null capsid had respective SEC A₂₆₀/A₂₈₀ values of 1.16, 1.10, and 1.01.The optimized AEX method used these materials to generate 6 or 7contiguous elution fractions each with SEC A₂₆₀/A₂₈₀≥1.25.

Loads containing 60%, 80%, and 100% null capsid had respective SECA₂₆₀/A₂₈₀ values of 0.90, 0.77, and 0.62. The optimized AEX methodenriched starting materials with 60%, 80%, and 100% null capsid togenerate elution fractions with maximum SEC A₂₆₀/A₂₈₀ values of 1.23,1.16, and 0.83, respectively.

The AAV9 upstream process, implemented at the 250 L and 2000 L singleuse bioreactor (SUB) scale, combined with downstream operations ofharvest and affinity chromatography produced AAV9 affinity eluate withSEC A₂₆₀/A₂₈₀ of 1.10±0.1, (n=7). Therefore, upstream and downstreamprocessing (including the optimized AEX process described here)generated AAV9 capsids with a % full that is useful for gene therapyapplications. Based on these results, the optimized AEX process wasscaled up to enable manufacturing at the 250 L and 2000 L SUB scale,described in the example that follows.

TABLE 17 Optimized AEX chromatography method performed with a POROS ™ 50HQ column on starting materials with a varying % of full capsids. Allruns involved uniformly challenging columns at 2.2 × 10¹⁴ VP/mL resinwith a varying % of null capsid transfection starting material. ColumnBed Height: 19.5 cm Load Challenge: Column Column Cross Sectional 2.2 ×10¹⁴ VP/mL of Resin Diameter: 0.66 cm Area: 0.34 cm² Residence StepColumn Volume = 6.67 mL Time Flow Rate Description Solution DescriptionCV (min/CV) (mL/min) Sanitization 0.5M NaOH 7.5 4 1.67 Regeneration 2MNaCl, 100 mM Tris pH 9 5 4 1.67 Equilibration 1 100 mM Tris, pH 9 5 41.67 Equilibration 2 500 mM sodium acetate, 100 mM Tris, 5 4 1.67 0.01%P188, pH 8.9 Equilibration 3 200 mM Histidine, 200 mM Tris, 0.5% 5 41.67 P188, pH 8.8 Sample Loading Affinity Eluate^(N), diluted 15X with0.5% 10 4 1.67 P188, 200 mM Histidine, 200 mM Tris pH 8.8; 0.2 μmfiltered Equilibration 4 100 mM Tris, 0.01% P188, pH 8.9 5 4 1.67 SodiumAcetate A - 100 mM Tris, 0.01% P188, pH 8.9 20 4 1.67 Gradient ElutionB - 500 mM NaAcetate, 100 mM Tris, 0.01% P188, pH 8.9 Gradient is runfrom 0-100% B over 20 CV Collect fractions of ⅓ CV starting at A₂₈₀ ≥10mAU, and ending at A₂₈₀ <10 mAU* Gradient Hold 500 mM NaAcetate, 100 mMTris, 0.01% 5 4 1.67 P188, pH 8.9 Sanitization 0.5M NaOH 7.5 4 1.67Regeneration 2M NaCl, 100 mM Tris pH 9 5 4 1.67 Equilibration 5 100 mMTris, pH 9 5 4 1.67 Storage 17% Ethanol 5 4 1.67 *A 2 mm path length UVmonitor was employed. NMixture of standard AAV9 affinity eluate and nullcapsid transfection AAV9 affinity eluate at volumetric ratios of 0%,20%, 40%, 60%, 80%, and 100% Null capsids.

TABLE 18 SEC A₂₆₀/A₂₈₀ of chromatographic fractions generated using theoptimized AEX method performed on feed materials with varying mixturesof % Null and a standard affinity pool. Mixture Load F/T 1 2 3 4 5 6 7 89 10 100% Null 0.62 0.58 0.83 0.83 0.81 0.78 0.74 0.68 0.63 0.61 0.610.61 80% Null 0.77 0.63 1.14 1.16 1.15 1.14 1.12 1.06 0.95 0.83 0.770.74 60% Null 0.90 0.68 1.20 1.23 1.22 1.22 1.21 1.17 1.09 0.98 0.900.84 40% Null 1.01 0.80 1.26 1.27 1.26 1.26 1.26 1.23 1.19 1.10 1.030.97 20% Null 1.10 0.85 1.26 1.28 1.28 1.29 1.28 1.26 1.24 1.18 1.131.09 0% Null 1.16 0.96 1.28 1.30 1.30 1.30 1.30 1.30 1.28 1.24 1.23 1.22Mixture Load F/T 11 12 13 14 100% Null 0.62 0.58 0.61 0.60 0.60 0.60 80%Null 0.77 0.63 0.71 0.68 0.66 0.65 60% Null 0.90 0.68 0.79 0.74 0.690.67 40% Null 1.01 0.80 0.92 0.84 0.79 0.75 20% Null 1.10 0.85 1.04 0.990.90 0.87 0% Null 1.16 0.96 1.17 1.13 1.04 1.00 F/T: flow through(unbound) fraction. Elution fractions are given as numbers. Ratios ≥1.25 are bolded.

Example 8: Scale-Up of Optimized AEX Process for Enrichment of Full AAV9Vectors

An optimized AEX method was scaled-up to enrich for full AAV9 vectorsfor downstream processing of AAV vectors produced in 250 L and 2000 Lsingle use bioreactors (SUBs). POROS™ 50 HQ columns were sized based ona scale-independent maximum challenge of 3×10¹⁴ VG/mL resin. Table 19and Table 20 provide optimized AEX methods implemented for the 250 L and2000 L SUB processes, respectively. At the 250 L SUB scale, a 1.3 L AEXcolumn, with a 10 cm inner diameter (ID) and a 16 cm bed height wasused. The AEX process was implemented at the 2000 L SUB scale using a6.4 L AEX column with a 20 cm ID and a 20.5 cm bed height. A residencetime of 4±0.5 minutes/CV was fixed across both scales, leading tovolumetric flow rates of 314 mL/min and 1.8 L/min for all steps withinthe AEX processes for the 250 L and 2000 L SUBs, respectively. Theseflow rates were in acceptable ranges to enable pumps and mixers onchromatography skids to form smoothly shaped linear sodium acetategradients during product elution. Across both scales, eachchromatography step used the same set of buffers for the same CV lengthwith the exception that the 2000 L SUB AEX process included Water ForInjection Flushes and pre-use sanitization and regeneration steps. FIG.10 and FIG. 11 provide chromatograms of representative AEX runs carriedout at the 250 L SUB scale and the 2000 L SUB scale. Across all scalestested (various small scale runs, 250 L and 2000 L SUB scales), theoptimized AEX process produced similar A260/A280 chromatographicprofiles.

At both the 250 L and 2000 L scales, elution fractions sized ⅓^(rd)column volume (CV) were collected. Fractions were neutralized with 250mM sodium citrate, pH 3.5 and assayed by various analytical techniques.SEC was carried out to determine the A260/A280 ratio and the percentageof high molar mass species (% HMMS). Residual amounts of host cellprotein (HCP) and host cell DNA (HC-DNA) were determined via ELISA andqPCR, respectively. At the 250 L scale, qPCR was used to measure ITRcopies to quantify the VG. At the 2000 L scale, qPCR was used to measuretransgene copies to quantify the VG.

TABLE 19 Optimized AEX chromatography method implemented at 250 L SUBscale on a 1.3 L POROS ™ 50 HQ column. Column Bed Height: 16 cm LoadChallenge: Column Column Cross Sectional 1.7 × 10¹³-5.3 × 10¹³ VG/mL ofResin Diameter: 10 cm Area: 78.5 cm² Residence Linear Flow Step ColumnVolume = 1256 mL Time Velocity Rate Description Solution Description CV(min/CV) (cm/hr) (mL/min) Equilibration 1 500 mM sodium acetate, 100 mM5 4 240 314 Tris, 0.01% P188, pH 8.9 Equilibration 2 200 mM Histidine,200 mM Tris, 0.5% 5 4 240 314 P188, pH 8.8 Sample Affinity Eluate,diluted 15X with 0.5% N/A 4 240 314 Loading P188, 200 mM Histidine, 200mM Tris pH 8.8; filtered through 0.2 μm filter Equilibration 3 100 mMTris, 0.01% P188, pH 8.9 5 4 240 314 Sodium Acetate A - 100 mM Tris,0.01% P188, pH 8.9 20 4 240 314 Gradient Elution B - 500 mM sodiumacetate, 100 mM Tris, 0.01% P188, pH 8.9 Run gradient from 0-100% B over20 CV Collect ≥10 fractions of ⅓ CV starting at A₂₈₀ ≥10 mAU (2 mm pathlength) Sanitization 0.5M NaOH 5 4 240 314 Regeneration 2M NaCl, 100 mMTris pH 9 5 4 240 314 Equilibration 4 100 mM Tris, pH 9 5 4 240 314Storage 17.5% Ethanol 3 4 240 314

TABLE 20 Optimized AEX chromatography method performed at 2000 L SUBscale on a 6.4 L POROS ™ 50 HQ column. Column Bed Height: 20.5 cm LoadChallenge*: Column Column Cross Sectional 2.7 × 10¹²-6.8 × 10¹³ VG/mL ofResin Diameter: 20 cm Area: 314 cm² Residence Linear Flow Step ColumnVolume = 6.4 L Time Velocity Rate Description Solution Description CV(min/CV) (cm/hr) (L/min) Pre-Use Flush Water for Injection (upward flow)5 3.6 300 1.8 Sanitization 1 0.5M NaOH (upward flow) 16 3.6 300 1.8Regeneration 1 2M NaCl, 100 mM Tris, pH 9 5 3.6 300 1.8 Equilibration 1100 mM Tris, pH 9 5 3.6 300 1.8 Equilibration 2 500 mM sodium acetate,100 mM 5 3.6 300 1.8 Tris, 0.01% P188, pH 8.9 Equilibration 3 200 mMHistidine, 200 mM Tris, 5 3.6 300 1.8 0.5% P188, pH 8.8 Product AffinityEluate, diluted 15X with 0.5% N/A 3.6 300 1.8 Loading P188, 200 mMHistidine, 200 mM Tris, pH 8.8; filtered through 0.2 μm filter (in-line)Equilibration 4 100 mM Tris, 0.01% P188, pH 8.9 5 3.6 300 1.8 SodiumAcetate A - 100 mM Tris, 0.01% P188, pH 8.9 20 3.6 300 1.8 GradientElution B - 500 mM sodium acetate, 100 mM Tris, 0.01% P188, pH 8.9Gradient is run from 0-100% B over 20 CV Collect 10 fractions of 1/3 CVstarting at A₂₈₀ ≥0.5-5 mAU/mm path length* Sanitization 2 0.5M NaOH(upward flow) 16 3.6 300 1.8 Regeneration 2 2M NaCl, 100 mM Tris pH 9 53.6 300 1.8 Equilibration 4 100 mM Tris, pH 9 5 3.6 300 1.8 Post-useWater for injection 5 3.6 300 1.8 flush Storage 17.5% Ethanol 3 3.6 3001.8 *Resin challenges were determined using a qPCR method that measuredtransgene copies.

Table 21 provides SEC A260/A280 ratios of elution fractions generatedwith the 250 L and 2000 L SUB AEX processes, respectively. The processdemonstrated robustness to a broad range of column challenge:2.7×10¹²-6.8×10¹³ VG/mL resin. Nine out of ten AEX runs produced atleast 6 fractions with SEC A₂₆₀/A₂₈₀ ratios≥1.25. Batch 250 L-1 used theaffinity pool with the lowest SEC ratio in the study (0.94) and was theonly AEX run that did not generate fractions with a ratio≥1.25. For eachAEX run, fraction 5 yielded the highest SEC A₂₆₀/A₂₈₀ ratio in 7 out ofthe 10 runs, thus showing high consistency in chromatography andfraction collection operations across two different scales and variousVG/mL resin challenges.

Table 22 and Table 23 provide impurity profiles, % HMMS, and SECA₂₆₀/A₂₈₀ of individual AEX fractions from the 250 L-4 and the 2000 L-4batches, respectively. The optimized AEX process cleared high amounts ofHCP from affinity pools. For instance, the 250 L-4 affinity poolcontained 51 pg HCP/1×10⁹ VG, and the optimized AEX process cleared theHCP to LLOQ in elution fractions 2-8, used to form the AEX pool. The2000 L-4 affinity pool contained 331 pg HCP/1×10⁹ VG that was cleared toLLOQ in elution fractions 2-9, used to form the AEX pool.

The AEX process does not significantly reduce HC-DNA levels. The AEXprocess, using the sodium acetate elution gradient, resolved HMMS. Earlyelution fractions were relatively depleted in HMMS (e.g., fractions 1-5in both 250 L-4 and 2000 L-4 runs contained <3% HMMS). Conversely, laterelution fractions contained higher relative levels of HMMS (e.g.,fractions 8-10 from the 2000 L-4 run contained >7% HMMS).

TABLE 21 SEC A₂₆₀/A₂₈₀ of affinity eluate pool and AEX chromatographyfractions generated at 250 L and 2000 L SUB scale. VG/mL Batch resinAffinity Fraction number Scale ID challenge Pool 1 2 3 4 5 6 7 8 9 10250 L 250 L-1 5.3 × 10¹³ 0.94 1.11 1.20 1.23 1.23 1.23 1.22 1.19 1.131.03 1.05 SUB; 250 L-2 4.7 × 10¹³ 0.99 1.20 1.26 1.27 1.27 1.27 1.261.27 1.18 1.14 1.18 1.3 L 250 L-3 3.0 × 10¹³ 1.25 1.29 1.30 1.31 1.321.33 1.32 1.32 1.31 1.33 1.34 AEX 250 L-4 1.7 × 10¹³ 1.15 1.21 1.27 1.291.30 1.30 1.30 1.28 1.24 1.19 1.17 CV 250 L-5 1.9 × 10¹³ 1.19 1.27 1.301.31 1.32 1.32 1.32 1.30 1.25 1.22 1.23 2000 L 2000 L-1 2.7 × 10¹² N/A1.20 1.25 1.27 1.27 1.28 1.27 1.31 1.22 1.18 1.14 SUB; 2000 L-2 1.1 ×10¹³ N/A 1.10 1.22 1.26 1.28 1.28 1.29 1.28 1.25 1.20 1.17 6.4 L 2000L-3 2.2 × 10¹³ 1.06 1.06 1.23 1.28 1.30 1.31 1.31 1.31 1.28 1.24 1.19AEX 2000 L-4 4.1 × 10¹³ 1.10 1.18 1.28 1.31 1.32 1.33 1.33 1.33 1.291.25 1.21 CV 2000 L-5 6.8 × 10¹³ 1.04 1.11 1.26 1.31 1.32 1.33 1.33 1.321.29 1.22 1.15 Elution fractions are given as numbers. Ratios ≥ 1.25 arebolded. 250 L SUB VG/mL resin challenges were determined using a qPCRmethod that measured ITR copies. 2000 L SUB VG/mL resin challenges weredetermined using a qPCR method that measured transgene copies.

TABLE 22 SEC A₂₆₀/A₂₈₀, % HMMS, and impurity profile of chromatographyfractions from an AEX run at the 250 L SUB scale (batch 250 L-4).Affinity Metric Pool 1 2 3 4 5 6 7 8 9 10 SEC A₂₆₀/A₂₈₀ 1.15 1.21 1.271.29 1.30 1.30 1.30 1.28 1.24 1.19 1.17 % HMMS 0.7 0.0 0.2 0.3 0.5 0.81.2 2.0 2.9 3.2 2.6 (SEC) % Purity — 99.2 99.4 99.3 99.3 99.3 99.2 99.199.1 99.3 99.2 (RP-HPLC) HC-DNA (pg)/ 4.0 1.9 5.0 5.7 3.4 1.8 1.3 1.21.3 1.2 0.6 1 × 10⁹ VG HCP (pg)/ 51.1 LLoQ LLoQ LLoQ LLoQ LLoQ LLoQ LLoQLLoQ LLoQ LLoQ 1 × 10⁹ VG Elution fractions are given as numbers.Elution fractions 2-8 had SEC A₂₆₀/A₂₈₀ ≥ 1.24 (bolded), and thus werepooled and processed forward. LLoQ—detected amount was lower than thelimit of quantitation of the assay.

TABLE 23 SEC A₂₆₀/A₂₈₀, % HMMS, and impurity profile of chromatographyfractions from an AEX run at the 2000 L SUB scale (batch 2000 L-4).Affinity Metric Pool 1 2 3 4 5 6 7 8 9 10 SEC A₂₆₀/A₂₈₀ 1.10 1.18  1.28 1.31  1.32  1.33  1.33  1.33  1.29  1.25 1.21 % HMMS 0.8 0  1.2  1.5 2.4 2.8 4.2 5.5 7.7 8.3 7.7 (SEC) % Purity — 79.6 97.6 98.5 98.7 98.8 98.4  97.7  97.2  97.0  98.0 (RP-HPLC) HC-DNA (pg)/ 9.1 26.5 20.2 15.012.0 9.3 6.5 4.7 2.7 4.0 5.8 1 × 10⁹ VG HCP (pg)/ 331 LLoQ LLoQ LLoQLLoQ LLoQ LLoQ LLoQ LLoQ LLoQ LLoQ 1 × 10⁹ VG Elution fractions aregiven as numbers. Elution fractions 2-9 had SEC A₂₆₀/A₂₈₀ ≥ 1.25(bolded), and thus were pooled and processed forward. LLoQ—detectedamount was lower that the limit of quantitation of the assay.

The 250 L AEX runs used various fraction pooling thresholds based on SECA₂₆₀/A₂₈₀ ratios. Fractions from 250 L AEX runs 250 L-1 and 250 L-4 werepooled based on where SEC A₂₆₀/A₂₈₀≥1.22 and≥1.24, respectively.Fractions from 250 L AEX runs 250 L-2, 250 L-3, and 250 L-5, and allfive 2000 L AEX runs were pooled based on where SEC A₂₆₀/A₂₈₀≥1.25.

Resulting AEX product pools from 2000 L batches 2000 L-2, 2000 L-3, 2000L-4 and 2000 L-5 were processed through a step of viral filtration while250 L batches and batch 2000 L-1 were not viral filtered. Resulting AEXpools and/or viral filtration pools were processed throughultrafiltration/diafiltration (UF/DF) and 0.2 μm filtration to generatedrug substance (DS). None of the steps after AEX chromatographysignificantly impacted AAV9 empty/full ratios. qPCR was performed onaffinity and AEX pools to determine % VG yield of the AEX step. Drugsubstance material was analyzed by AUC and SEC A260/A280 to determine %full of the purified AAV9 vector.

Table 24 reports results and shows the scaled-up AEX processes increased% full of the recovered AAV9 vector with high yields. The scaled-up AEXprocess enriched the % full of vectors to 45-65% of total capsids, andreduced the amount of empty capsids to ≤28% of total capsids in 9 out of10 drug substance batches, as measured by AUC. In all 10 runs, the AEXprocess increased SEC A260/A280 ratios in affinity pools from 0.94-1.25to 1.24-1.32 in drug substance. The average % VG step yield of the AEXprocess implemented at 250 L and 2000 L scales was 47+/−11%.

TABLE 24 AEX performance at 250 L and 2000 L SUB scale. AEX ProcessInputs Affinity AEX Process Outputs Pool DS¹ Analytical DS¹ VG/mL SECUltracentrifugation SEC % VG % VG % VG Batch resin A260/ % % % A260/Dilution Column Step ID challenge A280 full inter empty A280 Yield²Yield³ Yield⁴ 250 L 250 L-1 5.3 × 10¹³  0.94 45 19 37 1.24 92 40 37 SUB;250 L-2 4.7 × 10¹³  0.99 50 22 28 1.27 54 84 45 1.3 L 250 L-3 3.0 ×10¹³  1.25 65 25 10 1.32 73 82 60 AEX 250 L-4 1.7 × 10¹³  1.15 55 28 171.30 41 98 40 CV 250 L-5 1.9 × 10¹³  1.19 62 26 13 1.31 41 100 41 2000 L2000 L-1 2.7 × 10¹²* N/A 47 35 18 1.28 94 43 40 SUB; 2000 L-2 1.1 ×10¹³* N/A 45 37 18 1.29 154 20 31 6.4 L 2000 L-3 2.2 × 10¹³* 1.06 49 3120 1.30 88 61 54 AEX 2000 L-4 4.1 × 10¹³* 1.10 52 27 21 1.31 105 52 55CV 2000 L-5 6.8 × 10¹³* 1.04 50 28 22 1.30 137 48 66 average ± standard1.09 ± 0.10 52 ± 7 28 ± 5 20 ± 7 1.29 ± 0.02 88 ± 36 63 ± 26 47 ± 11deviation ¹‘DS’ indicates that AUC and SEC A₂₆₀/A₂₈₀ was performed onDrug substance (DS). None of the steps following AEX used to make drugsubstance impact AAV9 vector empty/full ratio. ²% VG Dilution Yield wascalculated as ((VG in diluted affinity pool)/(VG in affinity pool)) *100%. “Affinity pool” refers to material collected from an affinitycolumn; and is also referred to herein as an “affinity eluate.” ³% VGColumn Yield was calculated as ((VG in AEX pool)/(VG in diluted affinitypool)) * 100%. “Diluted affinity pool,” also referred to herein as a“diluted affinity eluate.” In this Example the diluted affinity eluatehas not been filtered. ⁴% VG Step Yield was calculated as ((VG in AEXpool)/(VG in affinity pool)) * 100%. 250 L SUB VG/mL resin challengesand % VG Yields were determined using a qPCR method that measured ITRcopies. ^(*)2000 L SUB VG/mL resin challenges and % VG Yields weredetermined using a qPCR method that measured transgene copies.

Example 9: Ultracentrifugation and Cation Exchange Chromatography forSeparation of Empty Capsids from Full AAV Vector Capsids

In another embodiment of the AAV9 vector 250 L SUB downstream process,density gradient ultracentrifugation (UC) was employed to separate emptycapsids from full vectors. Similar to a previously described method(Grieger et al. Molecular Therapy (2016) 24(2):287-297), bands thatcontained 40% and 60% iodixanol were formed in UC tubes. Affinityeluates containing 25% iodixanol were added to the UC tubes andultra-centrifuged. A fraction enriched in AAV capsids containing a fulllength vector genome was collected from the interface of the 40% and 60%iodixanol bands. To remove iodixanol, the fraction was diluted andloaded onto a cation exchange (CEX) chromatography column. AAV9 vectorcapsids bound to the CEX column, and the majority of iodixanol passedthrough the column in the unbound fraction. AAV9 vector capsids wereeluted from the CEX column in a fraction that was substantially free ofiodixanol. The CEX pool was processed forward through a UF/DF and 0.2 μmfiltration to generate drug substance (DS) comprising AAV9 vectorcapsids.

Table 25 provides process performance of the UC+CEX and the optimizedAEX methods and a comparison of resulting analytics on drug substancesproduced by these methods. The optimized AEX process, implemented atboth the 250 L and 2000 L scales, provided average VG yields of 45±8%and 50±13%, respectively. These values were higher than the average33±9% VG yield provided by the UC+CEX process. All three methodsproduced highly similar average DS readouts of SEC A₂₆₀/A280(1.26-1.30), % full (49-55%), and % empty (20-25%). The averagepercentage of intermediate capsids was slightly higher in DS produced bythe 2000 L AEX process (32±4%) as compared to DS produced by the 250 LAEX (24±3) and 250 L UC+CEX (23±4) processes.

The three methods produced DS with high similarity in % HMMS, % Purity,and levels of HCP and HC-DNA. Collectively, this data shows that the AEXprocess, implemented at the 250 L and 2000 L SUB scales, providedprocess performance and product quality highly similar to the 250 LUC+CEX process.

TABLE 25 Drug Substance characterization of AAV9 purified via downstreamprocesses that enriched for full vectors either via ultracentrifugationand cation exchange chromatography (UC + CEX), or the optimized AEXmethod. Capsid Separation Method UC + CEX AEX AEX Inputs SUB Scale 250 L250 L 2000 L No. of DS Batches  4 5 5 No. of Capsid Separation Runs (DS26 5 5 Sublots) Capsid Separation % VG Step Yield  33 ± 9 ^(α)   45 ± 8^(β)   50 ± 13 ^(β) (qPCR ^(γ)) Full A₂₆₀/A₂₈₀ (SEC) 1.26 ± 0.07  1.29 ±0.03  1.30 ± 0.01 Capsid % Full Capsids (AUC) 52 ± 11 55 ± 7 49 ± 2Characterization % Intermediate Capsids (AUC) 23 ± 4  24 ± 3 32 ± 4 %Empty Capsids (AUC) 25 ± 14  21 ± 10 20 ± 2 Impurity % HMMS (SEC) 3.4 ±0.9  2.6 ± 0.8  2.9 ± 0.4 Characte % Purity (RP-HPLC, Non-Reduced) 98.4± 1.3  98.6 ± 0.6 99.3 ± 0.3 Residual HCP (pg/1 × 10⁹ VG, LLOQ, < 2.6^(δ) LLOQ LLOQ ELISA) Residual HC-DNA (pg/1 × 10⁹ VG, 7.1 ± 3.4 17.4 ±6.7  9.3 ± 1.2 qPCR) ^(α) UC + CEX % VG step yield accounts for UC andCEX operations. ^(β) AEX % VG step yields account for both Dilution andAEX Chromatography operations. ^(γ) 250 L SUB % VG Step Yield wasdetermined using a qPCR method that measured ITR copies. 2000 L SUB % VGStep Yield was determined using a qPCR method that measured transgenecopies. ^(δ) Three UC + CEX drug substance lots had HCP levels that wereLLOQ, while one drug substance lot had 2.6 pg HCP/1 × 10⁹ VG. LLOQ-detected amount was lower that the limit of quantitation of the assay.

Example 10: Optimized AEX Process for Enrichment of Full AAV3B Vectors

HEK 293 cells were grown in suspension culture and transfected with 2plasmids to produce AAV3B vector per standard methods known in the art.HEK 293 cells were harvested, lysed, flocculated, and the resultinglysate was filtered. AAV3B vector was purified from the clarified lysateby affinity chromatography. An affinity column was equilibrated, loadedwith clarified lysate, washed, and the purified AAV3B vector was eluted.The AAV3B vector affinity pool (also referred to as affinity eluate) wasspiked with 25 mM MgCl₂ to achieve a final MgCl₂ concentration of about1.7 mM in the diluted affinity pool. The pH of the affinity pool was pH7.6. The affinity pool was diluted about 15-fold (14-fold to 17.8-folddepending on the run) with a buffer comprising 200 mM Histidine, 200 mMTris, 0.5% P188, pH 8.9. The resulting diluted affinity eluate had a pHof ≥8.6, and a conductivity of ≤2.5 mS/cm (target 2.3 mS/cm), and wasloaded on to an AEX column.

A POROS™ 50 HQ column with a 49 mL column volume, a bed height of cm anda diameter of 2.5 cm was used. The target column load challenge was1×10¹⁴ to 3×10¹⁴ vg/mL resin (e.g., about 2.4×10¹⁴ vg/mL). Table 26provides the optimized AEX method conditions. A residence time of 2minutes/CV was fixed for all steps within the AEX process to accommodatethe shallower elution gradient (as compared to previous Examples) andthe relatively smaller column. The elution gradient was 2.5 fold moreshallow than previous Examples in order to maximize empty/fullresolution. Empty capsids eluted first, followed by full AAV3B capsids(FIG. 12 ). In this Example, the full AAV3B capsids contained a vectorgenome with a transgene encoding the amino acid of SEQ ID NO:15 (coppertransporting ATPase 2 with deletion of metal binding sites 1-4).Consistent with the shallower gradient and broader elution peak, thefraction volume was increased to 0.5 CV (as opposed to 0.33 CV ofprevious Examples).

TABLE 26 Optimized AEX chromatography method for purification of fullrAAV3B vectors. Load Challenge: Bed Height: 10 cm 1-3E14 vg/mL resinDiameter: 2.5 cm Cross Sectional Area: 4.9 cm² Residence Linear StepColumn Volume = 49 mL Time Velocity Description Solution Description CVs(min/cv) (cm/hr) Sanitization 1 0.5M NaOH (downward flow) 8 2 298Regeneration 1 2M NaCl, 100 mM Tris, pH 9.0 5 2 298 Equilibration 10.01% P188, 500 mM sodium acetate 5 2 298 100 mM Tris pH 8.9Equilibration 2 0.5% P188, 200 mM histidine, 200 mM 5 2 298 Tris pH 8.8Load Affinity Pool, diluted with 0.5% P188, N/A 2 298 200 mM histidine,200mM Tris, pH 8.8 Equilibration 3 100 mM Tris, 0.01% P188, pH 8.9 5 2298 Sodium acetate A - 100 mM Tris, 0.01% P188, pH 8.9 37.5 2 298gradient elution B - 500 mM sodium acetate, 100 mM Tris, 0.01% P188, pH8.9 Gradient run from 0-75% over 37.5 CV Collected 20 fractions of 0.5CV from 32%-52% B into vessels Sanitization 2 0.5N NaOH (downward flow)8 2 298 Regeneration 2 2M NaCl, 100 mM Tris, pH 9.0 5 2 298Equilibration 4 100 mM Tris pH 9.0 5 2 298 Storage 17% ethanol 5 2 298

The gradient elution was run from 100% Buffer A to 25% Buffer A/75%Buffer B over 37.5 CV for a slope of 2% Buffer B/CV. When the percentageof Buffer B was 32% to 52% across the gradient, a total of 20 elutionfractions were collected. Fractions were collected into vesselspre-charged with 13.2% v/v (0.066 CV) of 250 mM sodium citrate, pH 3.5to neutralize the fraction and reduce exposure of the capsids toalkaline pH. The pH of the neutralized fractions ranged from pH 7.5 to7.7. The first elution fraction with an A260/A280≥0.98 (determined bySEC) was pooled with consecutive elution fractions, but no more than atotal of 11 factions (Table 27).

TABLE 27 Elution fractions 001 002 004 006 009 % vg A₂₆₀/ % vg A₂₆₀/ %vg A₂₆₀/ % vg A₂₆₀/ % vg A₂₆₀/ Batch per A₂₈₀ per A₂₈₀ per A₂₈₀ per A₂₈₀per A₂₈₀ No. load vg Ratio load vg Ratio load vg Ratio load vg Ratioload vg Ratio Fxn1 0.35% 0.62 0.34% 0.62 0.44% 0.62 1.02% 0.66 1.01%0.69 Fxn2 0.71% 0.65 0.78% 0.66 1.03% 0.67 1.99% 0.74 1.76% 0.77 Fxn30.15% 0.71 1.84% 0.75 2.09% 0.75 3.74% 0.86 3.05% 0.89 Fxn4 3.25% 0.813.89% 0.88 3.89% 0.88 6.52% 0.97 4.65% 0.99 Fxn5 4.29% 0.91 6.08% 1.016.28% 0.99 8.63% 1.05 6.44% 1.02 Fxn6 6.59% 0.99 8.81% 1.08 8.50% 1.0510.61%   1.08 7.34% 1.02 Fxn7 8.21% 1.00 9.88% 1.06 9.17% 1.03 7.70%1.06 7.07% 0.98 Fxn8 7.84% 0.97 9.71% 1.04 8.75% 0.98 8.88% 1.01 6.64%0.94 Fxn9 7.11% 0.92 8.02% 0.99 7.53% 0.91 6.83% 0.94 5.47% 0.91 Fxn104.79% 0.89 6.82% 0.95 5.29% 0.85 6.03% 0.87 4.42% 0.91 Fxn11 4.56% 0.875.51% 0.93 4.61% 0.81 4.55% 0.81 4.05% 0.91 Fxn12 4.42% 0.86 4.89% 0.913.73% 0.78 3.94% 0.78 3.45% 0.91 Fxn13 3.78% 0.86 3.99% 0.90 2.93% 0.772.58% 0.78 2.70% 0.90 Fxn14 2.96% 0.86 2.94% 0.91 2.33% 0.76 2.02% 0.772.14% 0.90 Fxn15 2.23% 0.85 2.21% 0.90 1.73% 0.75 1.66% 0.79 1.93% 0.91Fxn16 1.70% 0.85 1.76% 0.89 1.31% 0.81 1.08% 0.81 1.43% 0.91 Fxn17 1.15%0.85 1.21% 0.90 0.95% 0.82 0.50% 0.80 0.99% 0.93 Fxn18 0.78% 0.86 0.98%0.90 0.71% 0.84 0.51% 0.83 0.77% 0.92 Fxn19 0.57% 0.85 0.63% 0.88 0.46%0.80 0.33% 0.85 0.55% 0.93 Fxn20 0.42% 0.82 0.48% 0.84 0.32% 0.65 0.18%0.78 0.34% 0.90

Fractions in bold and underlined were pooled.

The pooled fractions were assayed by various analytical techniques. Theactual vg/mL resin challenge ranged from 6.3E13 to 9.4E13 with anaverage of 7.4E13±1.2E13. SEC was carried out to determine the A260/A280ratio. The A260/A280 of the AEX pool was increased in all runs ascompared to the A260/A280 of the affinity pool (Table 28). The percentfull, intermediate and empty capsids of the affinity pool and AEX elutepool were determined by analytical ultracentrifugation. Affinity pools,with an average percent full vectors of 11.2±2.1%, were enriched to22.9±2.9% full in the AEX pool. The same affinity pools were depleted ofempty capsids from 79.7±2.5% to 67.5±3.8% in the AEX pool.

The vg titer was determined by transgene QPCR (Table 28). The average %vg dilution yield was 120%±12%. The average % vg column yield was47%±11%.

TABLE 28 AEX performance characteristics for the purification of AAV3Bvectors. SEC A260/A280 Affinity Batch ID Pool AEX Pool SEC Δ(AEX-Affinity) 001 0.81 1.03 0.22 002 0.88 1.01 0.13 004 0.84 0.98 0.14006 0.87 0.97 0.10 009 0.86 1.01 0.15 AVG +/− StdDev 0.85 ± 0.025 1.00 ±0.022 0.15 ± 0.040

Δ % Full Affinity Pool AEX Pool (AEX- Batch ID % full % inter. % empty %full % inter. % empty Affinity) 001 7.2 12.9 79.9 18.7 9.9 71.4 11.50002 12.5 12.7 74.8 27.7 12.1 60.3 15.20 004 10.7 8.5 80.7 22.8 7.7 69.512.10 006 13.1 5.0 81.9 22.3 9.1 68.7 9.20 009 12.1 6.8 81.0 23.1 9.367.6 11.00 AVG +/− 11.1 ± 2.1 9.2 ± 3.2 79.7 ± 2.5 22.9 ± 2.9 9.6 ± 1.467.5 ± 3.8 11.8 ± 2.0 StdDev

VG Titer via qPCR VG/mL resin % VG Dilution % VG Column Batch IDChallenge Yield Yield 001 6.4E+13 113% 35% 002 7.1E+13 119% 67% 0046.3E+13 121% 45% 006 9.4E+13 141% 47% 009 8.0E+13 105% 42% AVG +/−StdDev 7.4E13 ± 1.2E13 120% ± 12% 47 ± 11%

The method described in this Example was used to produce purified rAAV3Bvector pools that were enriched for full capsids and depleted of emptycapsids as compared to the starting material, that is an affinityeluate.

We claim:
 1. A method of purifying an rAAV vector by AEX, the methodcomprising a step of: i) loading a solution comprising the rAAV vectorto be purified onto an AEX stationary phase in a column; ii) performinggradient elution of a material from the stationary phase in the columnwherein a percentage of a first gradient elution buffer is varied in amanner inversely proportional to variation in a percentage of a secondgradient elution buffer; and iii) collecting at least one fraction ofeluate from the column during the gradient elution beginning when theabsorbance of a column flow-through reaches an absorbance threshold, andwherein the at least one fraction of eluate comprises the rAAV vector tobe purified.
 2. The method of purifying a rAAV vector by AEX of claim 1,wherein the method further comprises measuring an absorbance of the atleast one fraction of eluate collected from the column and determiningan A260/A280 ratio.
 3. The method of purifying a rAAV vector by AEX ofclaim 1 or 2, wherein the solution comprising the rAAV vector to bepurified is diluted about 2-fold to 25-fold (e.g., 15-fold) with adilution solution comprising histidine, Tris and P188, and optionallyfiltered prior to application to the stationary phase.
 4. The method ofpurifying a rAAV vector by AEX of any one of claims 1-3, wherein thesolution is an affinity eluate.
 5. The method of purifying a rAAV vectorby AEX of claim 5, wherein a pH of the diluted, and optionally filteredaffinity eluate is increased as compared to a pH of the solution; andwherein a conductivity of the diluted, and optionally filtered affinityeluate is decreased as compared to a conductivity of the solution. 6.The method of purifying a rAAV vector by AEX of any one of claims 1-5,wherein the first gradient elution buffer comprises about 50 mM to about150 mM Tris, about 0.005% to about 0.015% P188 and has a pH of about pH8.5 to 9.5; wherein the second gradient elution buffer comprises about400 mM to about 600 mM sodium acetate, about 50 mM to about 150 mM Tris,about 0.005% to about 0.015% P188 and has a pH of about pH 8.5 to 9.5;and wherein 10 to 60 column volumes (CV) (e.g., about 20 CV, about 37.5CV) of the first gradient elution buffer, the second gradient elutionbuffer or a mixture of both are applied to the stationary phase duringthe gradient elution.
 7. The method of purifying a rAAV vector by AEX ofany one of claims 1-6, wherein at a start of the gradient elution thepercentage of the first gradient elution buffer is 50%-100% and at anend of the gradient elution the percentage of the second gradientelution buffer is 50%-100% and wherein the percentage of the secondelution buffer increases at a rate of about 2% to 5% per CV over thegradient elution.
 8. The method of purifying a rAAV vector by AEX of anyone of claims 1-7, wherein a concentration of sodium acetate of thefirst gradient elution buffer, the second gradient elution buffer or themixture of both increases continuously during the gradient elution; andwherein the concentration of the sodium acetate increases at a rate ofabout 10 mM/CV to 50 mM/CV (e.g., about 10 mM/CV, about 25 mM/CV) overthe gradient elution.
 9. The method of purifying a rAAV vector by AEX ofany one of claims 1-8, wherein full capsids are eluted from thestationary phase in a first elution peak and/or in a first portion of asecond elution peak during the gradient elution.
 10. The method ofpurifying a rAAV vector by AEX of any one of claims 1-9, wherein emptycapsids are recovered in the column flow-through, in a first elutionpeak and/or in a last portion of a second elution peak during thegradient elution.
 11. The method of purifying a rAAV vector by AEX ofany one of claims 1-10, wherein an absorbance of the at least onefraction of eluate is measured at 280 nm, and wherein optionally, anabsorbance threshold is ≥0.5 mAU/mm path length measured at 280 nm. 12.The method of purifying a rAAV vector by AEX of any one of claims 1-11,wherein a volume of the at least one fraction of eluate is equivalent to⅛ of a CV to 10 CV, e.g., ⅛ of a CV, ¼ of a CV, ⅓ of a CV, ½ of a CV, ¾of a CV, 1 CV, 2 CV, 3 CV, 4 CV, 5 CV, 6 CV, 7 CV, 8 CV, 9 CV, 10 CV ormore of a CV, and optionally, wherein an A260/A280 ratio of the at leastone fraction of eluate is ≥ to 1.25.
 13. The method of purifying a rAAVvector by AEX of any one of claims 1-12, wherein at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, ormore, fractions of eluate are collected.
 14. The method of purifying arAAV vector by AEX of any one of claims 1-13, wherein the method furthercomprises combining at least two fractions of eluate collected from thecolumn, each having an A260/A280 ratio of ≥0.98 or ≥1.0 to form a pooledeluate comprising the rAAV vector.
 15. The method of purifying a rAAVvector by AEX of claim 14, wherein the pooled eluate has a % VG columnyield of 20% to 100% (e.g., 63+/−26%), a % VG step yield of 31% to 66%(e.g., 47+/−11%) and/or an A260/A280 ratio of ≥1.0.
 16. The method ofpurifying a rAAV vector by AEX of claim 14 or 15, wherein the pooledeluate is enriched for full capsids, and/or depleted of empty capsids,as compared to the solution loaded onto the column.
 17. The method ofpurifying a rAAV vector by AEX of any one of claims 1-16, wherein apurified rAAV vector is produced.
 18. The method of purifying a rAAVvector by AEX of any one of claims 14-17, further comprising filteringthe pooled eluate by a method selected from the group consisting ofviral filtration, ultrafiltration/diafiltration (UF/DF), filtrationthrough a 0.2 μm filter and a combination thereof, to produce a drugsubstance.
 19. The method of purifying a rAAV vector by AEX of claim 18,wherein the drug substance comprises: i) 45% to 65% (e.g., 52+/−7%) fullcapsids of total capsids; ii) 19% to 37% (e.g., 28+/−5%) intermediatecapsids of total capsids; and/or iii) 10% to 37% (e.g., 20+/−7%) emptycapsids of total capsids.
 20. The method of purifying a rAAV vector byAEX of claim 18 or 19, wherein the drug substance is enriched for fullcapsids, and/or depleted of empty capsids, as compared to the solutionloaded onto the column.
 21. The method of purifying a rAAV vector by AEXof any one of claims 1-20, wherein the rAAV vector comprises an AAVcapsid protein from an AAV serotype selected from the group consistingof AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV10, AAVrh10, AAVrh74, AAV12, AAV2i8, NP4, NP22, NP66, AAVDJ, AAVDJ/8,AAVDJ/9, AAVLK03, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQID NO:5 of WO 2015/013313), RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4,RHM15-6, AAV hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV2i8,AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N,AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8,AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15.22. The method of purifying a rAAV vector by AEX of any one of claims1-21, wherein the rAAV vector comprises an AAV9 capsid protein and atransgene comprising the nucleic acid of SEQ ID NO:1.
 23. The method ofpurifying a rAAV vector by AEX of any one of claims 1-21, wherein therAAV vector comprises an AAV3B capsid protein and a transgene comprisinga nucleic acid encoding the amino acid sequence of SEQ ID NO:15.
 24. Amethod of preparing a solution comprising a rAAV vector for purificationby AEX, the method comprising a step of: i) diluting a first solution 2to 25-fold (e.g., 15-fold) with a dilution solution comprisinghistidine, Tris and P188; and optionally ii) filtering the firstsolution from step i) through a filter to produce a diluted, andoptionally filtered solution; wherein the pH of the diluted, andoptionally filtered solution is increased as compared to the pH of thefirst solution; and wherein the conductivity of the diluted, andoptionally filtered solution is decreased as compared to theconductivity of the first solution.
 25. The method of preparing asolution comprising a rAAV vector for purification by AEX of claim 24,wherein the first solution comprising the rAAV vector is selected fromthe group consisting of an affinity eluate, a supernatant from a celllysate and a post-harvest solution.
 26. The method of preparing asolution comprising a rAAV vector for purification by AEX of claim 24 or25, wherein the dilution solution comprises about 100 mM to 300 mM(e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g., about 200 mM)Tris, 0.1% to 1.0% (e.g., about 0.5%) P188 and has a pH of pH 8.5 to9.5.
 27. The method of preparing a solution comprising a rAAV vector forpurification by AEX of any one of claims 24-26, wherein i) a pH of thediluted, and optionally filtered solution is 8.5 to 9.5; ii) aconductivity of the diluted, and optionally filtered solution is 1.7 to3.3 mS/cm; and/or iii) a % VG dilution yield of the diluted solution is35% to 100%.
 28. The method of preparing a solution comprising a rAAVvector for purification by AEX of any one of claims 24-27, wherein therAAV vector comprises an AAV9 capsid protein or an AAV3B capsid protein.29. A purified rAAV vector prepared by a method comprising a step of: i)loading a solution comprising the rAAV vector to be purified onto an AEXstationary phase in a column; ii) performing gradient elution of amaterial from the stationary phase in the column wherein a firstgradient elution buffer, a second gradient elution buffer or a mixtureof both are applied to the stationary phase and a concentration of asalt is varied from 0 mM to 500 mM such that the rate of increase inconcentration of the salt over the course of the gradient elution isabout 10 mM/CV to 50 mM/CV (e.g., about 25 mM/CV); iii) collecting atleast one fraction of eluate from the column during gradient elutionbeginning when absorbance of a column flow-through reaches an absorbancethreshold; and/or vi) measuring an absorbance of the at least onefraction of eluate collected from the column and determining anA260/A280 ratio.
 30. The purified rAAV vector prepared by the method ofclaim 29, wherein the method further comprises combining at least twofractions of eluate collected from the column when the A260/A280 ratiois ≥1.0 to form a pooled eluate comprising the purified rAAV vector. 31.The purified rAAV vector prepared by the method of claim 29 or 30,wherein the salt is sodium acetate.
 32. The purified rAAV vectorprepared by the method of any one of claims 29-31, wherein the rAAVvector comprises an AAV9 capsid protein or an AAV3B capsid protein. 33.The purified rAAV vector prepared by the method of any one of claims29-32, wherein the solution comprising the rAAV vector is an affinityeluate that has been diluted and optionally filtered prior to loadingonto the stationary phase.
 34. The purified rAAV vector prepared by themethod of any one of claims 29-33, wherein the material eluted from thestationary phase comprises the rAAV vector.
 35. The purified rAAV vectorprepared by the method of any one of claims 30-34, wherein the methodfurther comprises filtering the pooled eluate by a method selected fromthe group consisting of viral filtration, ultrafiltration/diafiltration(UF/DF), filtration through a 0.2 μm filter and a combination thereof,to produce a drug substance.
 36. The purified rAAV vector prepared bythe method of claim 35, wherein the drug substance is used to make adrug product suitable for administration to a human subject to treat adisease, disorder or condition.
 37. The purified rAAV vector prepared bythe method of claim 36, wherein the disease, disorder or condition isDMD or Wilson's disease, and optionally wherein the rAAV vectorcomprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:2or SEQ ID NO:15.
 38. An solution comprising a rAAV vector forpurification by AEX prepared by a method comprising a step of: i)diluting an affinity eluate 2 to 25-fold (e.g., 15-fold) with a dilutionsolution comprising histidine, Tris, and P188; and optionally ii)filtering the affinity eluate from step i) through a 0.2 μm filter toproduce a diluted, and optionally filtered affinity eluate; wherein a pHof the diluted, and optionally filtered affinity eluate is increased ascompared to a pH of the affinity eluate; and wherein a conductivity ofthe diluted, and optionally filtered affinity eluate is decreased ascompared to a conductivity of the affinity eluate.
 39. The solutioncomprising a rAAV vector for purification by AEX prepared by a method ofclaim 38, wherein the dilution solution comprises about 100 mM to 300 mM(e.g., about 200 mM) histidine, 100 mM to 300 mM (about 200 mM) Tris,0.1% to 1.0% (about 0.5%) P188, pH 8.5 to 9.5.
 40. The solutioncomprising a rAAV vector for purification by AEX prepared by a method ofclaim 38 or 39, wherein the pH the affinity eluate is 3.0 to 4.4 priorto a step of diluting, and optionally filtering, and the pH of theaffinity eluate after the step of diluting, and optionally filtering, is8.5 to 9.5 or 8.7 to 9.0 (e.g., 8.8, 9.0).
 41. The solution comprising arAAV vector for purification by AEX prepared by a method of any one ofclaims 38-40, wherein the conductivity of the affinity eluate is 5.0 to7.0 mS/cm (e.g., 5.5 to 6.5 mS/cm) prior to the step of diluting, andoptionally filtering, and the conductivity of the affinity eluate afterthe step of diluting, and optionally filtering, is 1.7 to 3.3 mS/cm, 1.8to 2.8 mS/cm, and/or 2.2 to 2.6 mS/cm.
 42. The solution comprising arAAV vector for purification by AEX prepared by a method of any one ofclaims 38-41, wherein the % VG dilution yield of the diluted andoptionally filtered affinity eluate is 35% to 100%.
 43. The solutioncomprising a rAAV vector for purification by AEX prepared by a method ofany one of claims 38-42, wherein the rAAV vector comprises an AAV9 or anAAV3B capsid protein.
 44. The solution comprising a rAAV vector forpurification by AEX prepared by a method of any one of claims 38-43,wherein the diluted, and optionally filtered affinity eluate is loadedon an AEX stationary phase.
 45. A method of preparing a stationary phasefor use in a method of purifying a rAAV vector by AEX of any one ofclaims 1-23, the method comprising at least one step of: i) pre-useflushing comprising application of ≥4.5 CV (e.g., about 5 CV) of waterfor injection to an AEX stationary phase in a column; ii) sanitizingcomprising application of about 14.4 to 17.6 CV (e.g., about 16 CV) of asolution comprising about 0.1 M to 1.0 M NaOH to the AEX stationaryphase in the column, optionally by upward flow; and/or iii) regeneratingcomprising application of about 4.5 to 5.5 CV (e.g., about 5 CV) of asolution comprising about 1 M to 3 M NaCl, 50 mM to 150 mM Tris, pH 8.5to 9.5 to the AEX stationary phase in the column.
 46. A method ofregenerating an AEX stationary phase, the method comprising a step of:i) post-use sanitizing the stationary phase comprising application of14.4 to 17.6 CV (e.g., about 16 CV) of a solution comprising about 0.1 Mto 1.0 M NaOH to the stationary phase, optionally by upward flow; ii)regenerating the stationary phase comprising application of 4.5 to 5.5CV (e.g., about 5 CV) of a solution comprising about 1 M to 3 M NaCl, 50mM to 150 mM Tris, pH 8.5 to 9.5 to the stationary phase; iii)equilibrating the stationary phase comprising application of 4.5 to 5.5CV (e.g., about 5 CV) of a solution comprising about 50 mM to 150 mMTris, pH 8.5 to 9.5 to the stationary phase; iv) post-use flushing thestationary phase comprising application of ≥4.5 (e.g., about 5 CV) ofwater for injection to the stationary phase; and/or v) applying astorage solution to the stationary phase comprising application 2.7 to3.3 CV (e.g., about 3 CV) of a solution comprising about 17% to 17.5%ethanol to the stationary phase.
 47. The method of regenerating an AEXstationary phase of claim 46, wherein any one of steps i)-v) stepfollows a chromatography elution step of a method of purifying a rAAVvector by AEX.
 48. A regenerated AEX stationary phase prepared by amethod comprising a step of: i) post-use sanitizing of the stationaryphase comprising application of 14.4 to 17.6 CV (e.g., about 16 CV) of asolution comprising about 0.1 M to 1.0 M NaOH to the stationary phase,optionally by upward flow; ii) regenerating the stationary phasecomprising application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solutioncomprising about 1 M to 3 M NaCl, 50 mM to 150 mM Tris, pH 8.5 to 9.5 tothe stationary phase; iii) equilibrating the stationary phase comprisingapplication of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprisingabout 50 mM to 150 mM, Tris, pH 8.5 to 9.5 to the stationary phase; iv)post-use flushing of the stationary phase comprising application of ≥4.5(e.g., about 5 CV) of water for injection to the stationary phase;and/or v) applying a storage solution to the stationary phase comprisingapplication 2.7 to 3.3 CV (e.g., about 3 CV) of a solution comprisingabout 17% to 17.5% ethanol to the stationary phase.
 49. A regeneratedAEX stationary phase of claim 48, wherein the regenerated AEX stationaryphase is used for purification of a rAAV vector.
 50. A method ofpurifying an rAAV vector by AEX, the method comprising a step of: i)loading a solution comprising the rAAV vector to be purified onto an AEXstationary phase in a column; ii) performing gradient elution of amaterial from the stationary phase in the column wherein a percentage ofa first gradient elution buffer is varied in a manner inverselyproportional to variation in a percentage of a second gradient elutionbuffer; wherein at the start of the gradient elution the percentage ofthe first gradient elution buffer is about 75% to about 100% and at theend of the gradient elution the percentage of the second gradientelution buffer is about 60% to about 100%; and wherein the percentage ofthe second elution buffer increases at a rate of about 2% to 5% per CVover the gradient elution; iii) collecting at least one fraction ofeluate from the column when performing the gradient elution beginningwhen the percentage of the second gradient elution buffer is about 30%to about 35%, and wherein the at least one fraction of eluate comprisesthe rAAV vector to be purified.
 51. The method of purifying an rAAVvector by AEX of claim 50, wherein the solution comprising the rAAVvector is an affinity elutate that has been diluted about fold with abuffer comprising histidine, Tris and P188.
 52. The method of purifyingan rAAV vector by AEX of claim 50 or 51, wherein the first gradientelution buffer comprises 50 mM to 150 mM (e.g., about 100 mM) Tris,0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9)and/or the second gradient elution buffer comprises 400 mM to 600 mM(e.g., about 500 mM) sodium acetate, 50 mM to 150 mM (e.g., about 100mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5(e.g., pH 8.9).
 53. The method of purifying an rAAV vector by AEX of anyone of claims 50-52, wherein collecting at least one fraction of eluatefrom the column comprises collecting the at least one fraction of eluateinto a vessel comprising about 0.01 CV to 0.1 CV (e.g., about 0.066 CV)of a solution comprising 200 mM to 300 mM (e.g., about 250 mM) sodiumcitrate, pH 3.0 to 4.0 (e.g., about 3.5).
 54. The method of purifying arAAV vector by AEX of any one of claims 51-53, wherein the at least onefraction of eluate is enriched for full capsids, and/or depleted ofempty capsids, as compared to the affinity eluate; optionally whereinthe rAAV vector is a rAAV3B vector; and optionally wherein the AEXstationary phase is POROS™ 50 HQ.
 55. The method of purifying a rAAVvector by AEX of any one of claims 50-54, wherein the collecting atleast one fraction of eluate from the column when performing thegradient elution ends when the percentage of the second gradient elutionbuffer is about 50% to about 55%.